Personalized therapeutic approaches to prostate cancer

ABSTRACT

Methods and kits are provided for assessing the cancer risk of a subject having prostate cancer by detecting DNA copy number alterations. In particular, 16p13.3 gain, and further in addition PTEN deletion are assessed, and detected using probes such as FISH probes to provide a customized treatment options to the screened individual.

CROSS-REFERENCE TO PUBLICLY FOUNDED RESEARCH AND PREVIOUSLY-FILEDAPPLICATIONS

The present application claims priority from U.S. provisionalapplication 62/741,654 filed on Oct. 5, 2018 and herewith incorporatedin its entirety.

This invention was made with government support under W81XWH-11-1-0638awarded by the Department of Defense. The government has certain rightsin the invention.

FIELD OF THE INVENTION

The present disclosure relates to the use of genetic biomarkers forprognosing prostate cancer and personalizing therapeutic approaches.

BACKGROUND

Prostate cancer remains a major clinical burden, being the mostprevalent cancer and one of the leading causes of cancer-specific deathsin North American men. It is a clinically heterogeneous disease whereinthe majority of cancers display a favorable outcome, while a subsetaffecting a considerable number of patients progress to metastatic andlethal stage. Radical prostatectomy (RP) or radiation therapy isconsidered the standard primary treatment option for localized prostatecancer and more recently, active surveillance has emerged as a viablealternative for patients presenting favorable clinicopathologicfeatures.

One of the key challenges in the clinical management of prostate canceris to accurately distinguish indolent from aggressive tumors in order toavoid overtreatment of clinically insignificant cancers andundertreatment of tumors with metastatic potential. Serumprostate-specific antigen (PSA) levels, biopsy Gleason grade (GS) andclinical tumor stage (cT-stage) are used to risk stratify patients, butare not sufficient to accurately predict individual clinical outcome.Assessing the Gleason grade based on prostate biopsies is challengingand frequently leads to an underestimation of the actual grade of theentire tumor burden. In particular, precisely assessing GS on biopsiesis limited by the fact that partial sampling may result in anunderestimation of the final score of cancer in the RP specimen. Themajority of patients undergoing RP present low-intermediate riskclinical features, and accurate prognosis within this subgroup ofpatients still remains a clinical challenge. Moreover, although mostpatients respond well to RP, a significant proportion will experience adisease recurrence, as assessed by a rise in serum PSA that mighteventually progress to the metastatic stage. Early identification andmore accurate risk stratification may, therefore, allow patients withaggressive tumors to receive appropriate treatment without delay whilesparing patients with clinically favorable tumors from treatment sideeffects.

To address the shortcomings of the clinicopathologic predictors and tobetter capture the clinical heterogeneity of prostate cancer, improvedbiomarkers are needed.

SUMMARY

The present application concerns methods for prognosing and treatingprostate cancer using 16p13.3 and/or 10q23.3 (PTEN) biomarkers.

In a first aspect, the present disclosure provides a method ofprognosing a subject with prostate cancer, the method comprising: a)providing a first sample from the prostate from the subject suspected ofcomprising cancer cells; b) contacting the first sample with a firstprobe capable of specifically recognizing a 16p13.3 chromosome region,wherein the first probe is associated with a first label; c) contactingthe first sample with a first reference probe capable of specificallyrecognizing a reference region of chromosome 16, wherein the firstreference probe is associated with a first reference label; d) detectingthe signal from the first label and from the first reference label; ande) classifying the cancer risk of the subject based on the presence orabsence of a 16p13.3 gain, wherein the 16p13.3 gain is detected if thesignal from the first label of the first probe is greater than thesignal from the first reference label of the first reference probe. Inone embodiment, the method further comprises f) providing a secondsample from the prostate from the subject suspected of comprising cancercells; g) contacting the second sample with a second probe capable ofspecifically recognizing a 10q23.3 (PTEN) chromosome region, wherein thesecond probe is associated with a second label; h) contacting the secondsample with a second reference probe capable of specifically recognizinga reference region of chromosome 10, wherein the second reference probeis associated with a second reference label; i) detecting the signalfrom the second label and from the second reference label; and j)classifying the cancer risk of the subject based on the presence orabsence of a PTEN deletion, wherein the PTEN deletion is detected if thesignal from the second label is less than the signal from the secondreference label.

In a second aspect, the present disclosure provides a method ofdetermining the presence or absence of aggressive prostate cancer in asubject, the method comprising: a) providing a first sample from theprostate from the subject suspected of comprising cancer cells; b)contacting the first sample with a first probe capable of specificallyrecognizing a 16p13.3 chromosome region, wherein the first probe isassociated with a first label; c) contacting the first sample with afirst reference probe capable of specifically recognizing a referenceregion of chromosome 16, wherein the first reference probe is associatedwith a first reference label; d) detecting the signal from the firstlabel and from the first reference label; and e) characterizing theprostate cancer aggressiveness of the subject based on the presence orabsence of a 16p13.3 gain, wherein the 16p13.3 gain is detected if thesignal from the first label is greater than the signal from the firstreference label, and the subject is characterized as having theaggressive prostate cancer in the presence of the 16p13.3 gain. In oneembodiment, the method further comprises f) providing a second samplefrom the prostate from the subject suspected of comprising cancer calls;g) contacting the second sample with a second probe capable ofspecifically recognizing a 10q23.3 (PTEN) chromosome region, wherein thesecond probe is associated with a second label; h) contacting the secondsample with a second reference probe capable of specifically recognizinga reference region of chromosome 10, wherein the second reference probeis associated with a second reference label; i) detecting the signalfrom the second label and from the second reference label; and j)characterizing the prostate cancer aggressiveness of the subject basedon the presence or absence of a PTEN deletion, wherein the PTEN deletionis detected if the signal from the second label is less than the signalfrom the second reference label, and the subject is characterized ashaving the aggressive prostate cancer in the presence of the PTENdeletion.

In a third aspect, the present disclosure provides a method ofdetermining recurrence-free and/or metastasis-free survival in a subjecthaving prostate cancer, the method comprising: a) providing a firstsample of the prostate from the subject suspected of comprising cancercells; b) contacting the first sample with a first probe capable ofspecifically recognizing a 16p13.3 chromosome region, wherein the firstprobe is associated with a first label; c) contacting the first samplewith a first reference probe capable of specifically recognizing areference region of chromosome 16, wherein the first reference probe isassociated with a first reference label; d) detecting the signal fromthe first label and from the first reference label; and e) determiningthe recurrence-free or metastasis-free survival of the subject based onthe presence or absence of a 16p13.3 gain, wherein the 16p13.3 gain isdetected if the signal from the first label of the first probe isgreater than the signal from the first reference label of the firstreference probe, and the subject is characterized as having a reducedthe recurrence-free or metastasis-free survival in the presence of the16p13.3 gain. In one embodiment, the method further comprises f)providing a second sample from the prostate from the subject suspectedof comprising cancer cells; g) contacting the second sample with asecond probe capable of specifically recognizing a 10q23.3 (PTEN)chromosome region, wherein the second probe is associated with a secondlabel; h) contacting the second sample with a second reference probecapable of specifically recognizing a reference region of chromosome 10,wherein the second reference probe is associated with a second referencelabel; i) detecting the signal from the second label and from the secondreference label; and j) determining the recurrence-free ormetastasis-free survival of the subject based on the presence or absenceof a PTEN deletion, wherein the PTEN deletion is detected if the signalfrom the second label is less than the signal from the second referencelabel, and the subject is characterized as having a reduced therecurrence-free or metastasis-free survival in the presence of the PTENdeletion.

In some embodiments, the sample is a tumor sample. In one embodiment,the first sample is the second sample. In some embodiments, the subjectpresents low to intermediate risk clinical features. In someembodiments, the first probe is derived from a RP11-20I23 DNA clone,variants thereof, fragments thereof, or complements thereof. While inother embodiments, the first probe is capable of hybridizing to one ormore genes of the 16p13.3 chromosome region comprising PKD1, RAB26,TRAF7, CASKIN1, MLST8, PGP, E4F1, DNASE1L2, ECI1, RNPS1, ABCA3, ABCA17A,CCNF, NTN3, TBC1D24, ATP6VOC, AMDHD2, CEMP1, PDPK1, variants thereof,fragments thereof, or complements thereof. In some embodiments, thefirst reference probe is capable of specifically recognizing a 16qhcentromeric region. In one embodiment, the first reference probe is aderived from a pHuR-195 DNA clone, variants thereof, fragments thereof,or complements thereof. In some embodiments, the second probe is derivedfrom a CTD-2557P6 DNA clone, variants thereof, fragments thereof, orcomplements thereof. In some embodiments, the second reference label isassociated with a second reference probe capable of specificallyrecognizing a 10p11.1-q11.1 centromeric region. In one embodiment, thesecond reference probe is a CEP10 probe, variants thereof, fragmentsthereof, or complements thereof. In some embodiments, the probes arecovalently bonded to the labels. In some embodiments, the labels arefluorescent labels. In one embodiment, the fluorescent labels aredetected with fluorescent in situ hybridization (FISH) method. In someembodiments, the method comprises classifying the tumor of the subjectprior to step (a). In some embodiments, the method further comprisestreating the subject with an adjuvant or a neoadjuvant therapy if thesubject is characterized as being associated with poor prognosis. Insome embodiments, the adjuvant or neoadjuvant therapy comprises surgery,radiation therapy, hormonotherapy and/or chemotherapy. In someembodiments, the subject is a human. In some embodiments, the subjecthas previously had a radical prostatectomy, and in one embodiment, thesample is from a resected tissue. In some embodiments, the sample isfrom a biopsy.

In a fourth aspect, the present disclosure provides a method of treatinga subject having prostate cancer with an adjuvant or a neoadjuvanttherapy, the method comprising: a) providing a tumor sample of theprostate cancer from the subject; b) performing the method of any one ofclaims 1 to 26 on the sample to determine if the subject has a 16p13.3gain and optionally a PTEN deletion; c) characterizing the prognosis ofthe subject; and d) if the subject has a poor prognosis, administeringan adjuvant or neoadjuvant therapy to the subject. In some embodiments,the adjuvant or neoadjuvant therapy surgery, radiation therapy,hormonotherapy and/or chemotherapy.

In a fifth aspect, the present disclosure provides a kit for theassessment of a cancer prognosis in a subject suspected of having orhaving prostate cancer, the kit comprising: a) a first probe capable ofspecifically recognizing a 16p13.3 chromosome region, wherein the firstprobe is associated with a first label; and b) a first reference probecapable of specifically recognizing a reference region of chromosome 16,wherein the first reference probe is associated with a first referencelabel. In one embodiment, the kit further comprises c) a second probecapable of specifically recognizing a 10q23.3 (PTEN) chromosome region,wherein the second probe is associated with a second label; and d) asecond reference probe capable of specifically recognizing a referenceregion of chromosome 10, wherein the second reference probe isassociated with a second reference label.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus generally described the nature of the invention, referencewill now be made to the accompanying drawings and tables, showing by wayof illustration, a preferred embodiment thereof, and in which:

FIG. 1A to 1D show a dual-color FISH analysis of 16p13.3 gain in FFPEprostate cancer specimens. All representative pictures were taken at X96magnification.

FIG. 1A The arrows indicate, normal interphase nuclei with 2 orangesignals (16p13.3 locus) and 2 green signals (centromere 16) in the tumorspecimen with no 16p13.3 gain.

FIG. 1B The arrows indicate nuclei with 3 orange signals (16p13.3 locus)and 2 green signals (centromere 16) per nucleus, indicating a singlecopy 16p13.3 gain in a prostate tumor.

FIG. 1C The arrows indicate nuclei with high level of gain (>3 orangesignals and 2 green signals) in a prostate tumor.

FIG. 1D The pie chart represents the proportion of RP specimens inMcGill cohort, harboring 16p13.3 gain as assessed by FISH.

FIG. 2A to 2E show prognostic value of the 16p13.3 genomic gain inprimary tumors of prostate cancer patients. Kaplan-Meier recurrence-freesurvival analysis of prostate cancer patients stratified on the basis of16p13.3 gain status determined by FISH. Number of patients at risk atrespective time points and P value (log-rank test) are indicated.

FIG. 2A All RP patients with clinical follow-up for BCR (n=238)

FIG. 2B The subgroup of patients belonging to low-intermediate riskpatients with PSA≤10 (n=189).

FIG. 2C Patients with GS≤7 (C; n=222).

FIG. 2D Patients with stage T2 (n=164).

FIG. 2E Kaplan-Meier metastases-free survival analysis of all casesstratified on the basis of 16p13.3 gain status (n=257).

FIG. 3A to 3D show improved risk stratification upon combination of16p13.3 gain and standard prognostic markers. Number of patients at riskat respective time points and P value (log-rank test) are indicated. The16p13.3 gain status was combined with individual standardclinicopathologic variables:

FIG. 3A PSA (≤10 vs. >10 ng/mL);

FIG. 3B GS (≤GS7 vs. ≥8); and

FIG. 3C pT-stage (pT2 vs. pT3) and cases with zero, one or two positivemarkers were compared by Kaplan-Meier analyses.

FIG. 3D The 16p13.3 gain status and all the 3 clinico-pathologicvariables described above were used to stratify patients and cases withzero, one, two, three, or all four positive markers were grouped tocompare BCR outcome.

FIG. 4A to 4C show 16p13.3 gain status affords additional prognosticinformation to the preestablished CAPRA-S score risk groups. Number ofpatients at risk at respective time points and P value (log-rank test)are indicated.

FIG. 4A Kaplan-Meier curves validating the prognostic significance ofCAPRA-S risk groups when stratified as low risk (LR, CAPRA-S score 0-2),intermediate risk (IR, CAPRA-S score 3-5), and high risk (HR, CAPRA-Sscore≥6).

FIG. 4B Each CAPRA-S risk group was further subdivided on the basis ofthe presence (+) or absence (−) of the 16p13.3 gain.

FIG. 4C Four risk groups were derived by merging the groups with theoverlapping risk of BCR from FIG. 4B as follows: LR^(+/−); IR⁻; IR⁺ orHR⁻; and HR⁺; respectively.

FIGS. 5A and 5B show 16p13.3 gain status affords additional prognosticinformation to the pre-established CAPRA-S score risk groups in anindependent dataset from Taylor et al. Number of patients at risk atrespective time points and P-value (log-rank test) are indicated.

FIG. 5A Kaplan-Meier curves of CAPRA-S risk groups when stratified asLow Risk (LR, CAPRA-S score 0-2), Intermediate Risk (IR, CAPRA-S score3-5) and High Risk (HR, CAPRA-S score ≥6).

FIG. 5B The four risk groups incorporating the CAPRA-S score with thepresence (+) or absence (−) of the 16p13.3 gain as follows: LR^(+/−);IR⁻; IR⁺ or HR⁻; and HR⁺; respectively.

FIG. 6A to 6D show dual-color FISH analysis of PTEN (10q23) deletion inprostate cancer specimens.

FIG. 6A The arrows show normal interphase nuclei with 2 green and 2orange signals in a PCa tumor with no PTEN deletion.

FIG. 6B The arrows show 2 green and 1 orange signals in a tumorharboring hemizygous PTEN deletion.

FIG. 6C The arrow shows 2 green and 0 orange signals in a homozygousPTEN-deleted case. FISH analysis.

FIG. 6D The pie chart represents detected hemizygous in 80/287 (28%),homozygous in 17/287 (6%), and no PTEN deletion in 189/287 (66%) of theprimary radical prostatectomy samples on the McGill urology tissuemicroarray (n=287).

FIG. 7A to 7H show prognostic value of the PTEN genomic deletion inprostate tumors. Kaplan-Meier recurrence-free survival analysis ofpatients stratified on the basis of PTEN deletion status determined byFISH. Censored data (tick marks), number of patients at risk atrespective time points, and P-value (log-rank test) are indicated. Theresults are shown in:

FIG. 7A All radical prostatectomy patients with clinical follow-up forbiochemical recurrence;

FIG. 7B Gleason grade group 1-2 (≤3+4),

FIG. 7C Gleason grade group 1-2 (≤3+4), stage pT2 and prostate-specificantigen ≤10 patients;

FIG. 7D Gleason grade group 2 (3+4);

FIG. 7E Gleason grade group 2 (3+4), stage pT2 and prostate-specificantigen ≤10 patients;

FIG. 7F low CAPRA-S score (0-2) risk patients; and

FIG. 7G intermediate CAPRA-S score (Klotz et al., 2014; Chang et al.,2012; Epstein et al., 2012) risk patients.

FIG. 7H Survival analysis based on PTEN deletion status shows worsemetastases-free survival.

FIG. 8A to 8D show improved risk stratification upon combination of16p13.3 gain and PTEN genomic deletion. Censored data (tick marks),number of patients at risk at respective time points, and P-value(log-rank test) are indicated. Kaplan-Meier recurrence-free survivalanalysis of prostate cancer patients stratified on the basis of

FIG. 8A PTEN deletion status in cases harboring no 16p13 gain;

FIG. 8B 16p13 gain status in cases harboring no PTEN deletion; and

FIG. 8C in patients with none, either or both genomic alterations.

FIG. 8D Harrel C-index is improved when combining 16p13 gain, PTENdeletion and CAPRA-S score risk group, compared to individual markers oreither of the three.

DETAILED DESCRIPTION

Genomic profiling studies have highlighted disease heterogeneity, and inparticular DNA copy number alterations (CNA) are common in cancer andhave been associated with molecular subtypes of prostate cancer,supporting the existence of alternative parallel pathways oftumorigenesis.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, melanoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small-cell lung cancer, adenocarcinoma ofthe lung, squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidneycancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, and various types of head and neck cancer. As usedherein, “prostate cancer” refers to cancer in the prostate gland in themale reproductive system, and may include cancer in the surroundinglymph nodes as well as metastatic tumors. As used herein,“prostatectomy” refers to the surgical removal of all or part of theprostate gland and surrounding tissue.

By “nucleic acid” is meant to include any DNA or RNA, for example,chromosomal, mitochondrial, viral and/or bacterial nucleic acid presentin tissue sample. The term “nucleic acid” encompasses either or bothstrands of a double stranded nucleic acid molecule and includes anyfragment or portion of an intact nucleic acid molecule.

By “gene” is meant any nucleic acid sequence or portion thereof with afunctional role in encoding or transcribing an RNA (rRNA, tRNA, or mRNA,the latter capable of 15 translation as a protein) or regulating othergene expression. The gene may consist of all the nucleic acidsresponsible for encoding a functional protein or only a portion of thenucleic acids responsible for encoding or expressing a protein. Thenucleic acid sequence may contain a genetic abnormality within exons,introns, initiation or termination regions, promoter sequences, otherregulatory sequences or unique adjacent regions to the gene.

In some embodiments, methods of prognosing a subject with prostatecancer are provided. As used herein, “prognosis” refers to theprediction of the likely or expected development of a disease, such ascancer, and includes a prediction of whether the signs and symptoms willimprove, worsen, or remain stable over time; life expectancy; presenceand number of metastasis; life quality expectations; potentialcomplications; and likelihood of survival. A poor prognosis isassociated with the development of the disease, the worsening of thesymptoms, a reduction in life expectancy; the presence or development ofmetastasis, the worsening of life quality, the presence of complication,a more aggressive cancer, and/or the reduction of survival (includingrecurrence-free and survival-free).

In some embodiments, methods of determining the presence or absence ofaggressive prostate cancer in a subject is provided. As used herein,“aggressive cancer” refers to tumor that forms, grows, or spreadsquickly, and/or fails to respond to therapy (medication or radiation orboth).

In some embodiments, methods of predicting recurrence-free and/ormetastasis-free survival in a subject having prostate cancer. The term“recurrence-free survival” refers to the length of time after primarytreatment for a cancer ends that the patient survives without any signsor symptoms (for example, the rise of PSA level) of that cancer. Forexample, the recurrence-free survival can be the time from primarytreatment to the first radiologically detected metastasis. The term“metastases-free survival” refers to time from the time interval fromprostate-specific antigen (PSA) recurrence to first radiographicallydetected metastasis.

In some embodiments, the subjects have not been previously diagnosedwith prostate cancer. In such embodiments, the subjects may haveelevated PSA levels and the methods can be used to determine if furtherinvestigation or treatment is needed or if only active surveillance iswarranted. In some alternative embodiments, the subjects may have beenpreviously diagnosed with prostate cancer. In such embodiments, some ofthe subjects may not have received primary treatment and the methods canbe used to determine if therapy is warranted or active surveillance ispreferred. Alternatively, the subjects may have received primarytreatment, such as radical prostatectomy or radiation therapy and themethods can be used to determine if additional therapy is warranted(hormonotherapy and/or chemotherapy for example) or if an activesurveillance is preferred.

In some embodiments, subjects have prostate-specific antigen (PSA) whichare detected in serum samples. In some embodiments, the methods of thepresent disclosure include determining the level of serum PSA ofsubjects and taking this information to determine the cancer risk, thepredisposition to aggressive cancer and/or to determine survivallikelihood. PSA level can be used to stratify the individuals accordingto the methods disclosed herein. In some embodiments, the subjects canbe classified as low-intermediate risk using standard clinicopathologicprognostic markers such as PSA. In some embodiments, the subjects withprostate cancer are classified as high risk using standardclinicopathologic prognostic markers such as PSA.

The tumors of subjects suspected of having or having been diagnosed withprostate cancer can be classified according to the Gleason score. Insome embodiments, the methods of the present disclosure includedetermining the Gleason score of the tumor of the subjects and takingthis information to determine the cancer risk, predisposition toaggressive cancer and/or to determine likelihood of survival. Grading atumor using the Gleason score is difficult. Usually, individuals scoring7 or higher are treated (with surgery or radiotherapy) whereasindividuals scoring 6 or lower are actively monitored. The methodsdescribed herein may be particularly useful if the subject scores a 6 orlower on the Gleason score as it may provide important information todetermine if a more aggressive therapy is warranted or if activesurveillance is more appropriate.

The methods described herein can be repeated in time (monthly, yearlyfor example) to determine if the prostate cancer evolves or remainsstable.

By “sample”, it is meant a collection of cells from a prostate suspectedof being cancerous. The sample can be, for example, a biopsy or aresected tissue obtained with surgery. The sample can be derived fromepithelium tissue; connective tissues, including blood vessels, bone andcartilage; muscle tissue; or nerve tissue. In one embodiment, the tumorsample is obtained from prostate gland tissue. The source of the samplemaybe solid tissue as from a fresh, frozen and/or preserved organ ortissue sample or biopsy or aspirate; blood or any blood constituents;bodily fluids such as cerebral spinal fluid, amniotic fluid, peritonealfluid, or interstitial fluid of the subject. The sample may also beprimary or cultured cells. The sample may contain compounds which arenot naturally intermixed with the tissue in nature such aspreservatives, anticoagulants, buffers, fixatives, nutrients,antibiotics, or the like. By “tumor sample”, it is meant a collection ofcells obtained from a cancerous tissue (such as a cancerous prostate) ofa subject or patient, in which some or all of the collection of cellsexhibit unregulated cancer cell growth. The tumor sample can be obtainedfrom the primary tumor or a tumor metastasis or both.

For the purposes herein a “section” of a tumor sample is meant a singlepart or piece of a tumor sample, e.g., a thin slice of tissue or cellscut from a tumor sample. It is understood that multiple sections oftumor samples may be taken and subjected to analysis according to thepresent disclosure, provided that it is understood that the presentdisclosure comprises a method whereby the same section of tissue samplemay be analyzed at both morphological and molecular levels, or may beanalyzed with respect to both protein and nucleic acid content. In anembodiment, the tumor sample is modified prior to the detection of thegenetic markers. For example, the tumor sample can be fixed prior to thedetection of the genetic markers.

As presented in the examples, it was shown that the 16p13.3 genomic gainis a predictor of poor clinical outcome in prostate cancer. Accordingly,detection of a 16p13.3 genomic gain can aid in better risk stratifyingsubjects when combined with standard clinicopathologic prognosticmarkers. As presented in the examples, it was also shown that the PTENgenomic deletion is a strong independent predictor of poor clinicaloutcome, including in low-intermediate risk patients and showed that thecombination of the PTEN deletion and the 16p13.3 gain status improvedpatient risk stratification.

As used herein, “genomic gain” or “gain” is an amplification of asection of DNA in the context of cancer where a tumor exhibits copynumber gain at different loci when compared to a normal/healthy sample.While “genomic deletion” or “deletion” refers to where a tumor exhibitscopy number loss at different loci when compared to a normal/healthysample. Both of these terms are referred to as copy number alterations(CNAs).

The present disclosure also provides methods of treating a subjecthaving prostate cancer is provided, with an adjuvant or neoadjuvanttherapy. “Treatment” refers to both therapeutic treatment andprophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those in which thedisorder is to be prevented. The term adjuvant therapy refers toadditional cancer treatment given after the primary treatment to lowerthe risk that the cancer will come back. Adjuvant therapy may includechemotherapy, radiation therapy, hormone therapy, targeted therapy, orbiological therapy. The term neoadjuvant therapy refers to a treatmentgiven as a first step to shrink a tumor before the main treatment, whichis usually surgery, is given. Examples of neoadjuvant therapy alsoinclude chemotherapy, radiation therapy, and hormonal therapy.

Probes for Determining Copy Number Alterations.

In the context of the present disclosure, samples (which can be tumorsamples) obtained from a tissue such as the prostate (which can be froma tumor located in the prostate or a metastasis which originated fromthe prostate) of a subject are analyzed for chromosomal abnormalities,including DNA copy number alterations (CNA). In some embodiments, thesamples are fixed to preserve the samples from decay due to autolysis orputrefaction. In some embodiments, samples are fixed using chemicalfixatives, such as formaldehyde, glutaraldehyde, alcohols, mercurials,or picrates. In one embodiment, the tumor sample are formalin-fixedparaffin-embedded (FFPE). In some embodiments, the tumor samples aresliced into tissue sections for analysis.

In some embodiments, a DNA CNA is detected using labeled probes,preferably fluorescent labeled probes and detected using florescencemicroscopy. In some embodiments, labels are provided separately fromtarget specific probes and binds to the probes for specific detection ofa target DNA sequence. In other embodiments, labels are bonded to targetspecific probes for specific detection of a target DNA sequence.

As used herein, the term “probe” or “hybridization probe” is a fragmentof DNA or RNA of variable length that specifically hybridizes to agenetic target to detect the presence of a target nucleotide sequencethat is complementary to the sequence in the probe. The probe can belabeled. The probe is at least 10, 100 or 1 000 nucleotides long and, insome embodiments, can span several thousand nucleotides. The probe canspan the genetic target or can be smaller than the genetic target. Acollection of probes overlapping or non-overlapping probes can also beused. As used herein, the term “hybridization” refers to annealing of asingle-stranded nucleic acid to a complementary nucleic acid. A probewhich is capable of hybridizing to a complementary target sequence doesnot need to be 100% complementary to the complementary sequence. In someembodiments, the probe is at least 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 96, 97, 98 or 99% complementary to the complementary targetsequence. Preferably such hybridization between the probe and itscomplementary nucleic acid sequence is specific, i.e., it occurs underhigh stringency conditions.

The term “label” when used herein refers to a compound or compositionwhich is bonded or fused directly or indirectly to a reagent, or bindsto a reagent, such as a nucleic acid probe or an antibody andfacilitates detection of the reagent to which it is conjugated or fused.The label may itself be detectable (e.g., radioisotope labels orfluorescent labels) or, in the case of an enzymatic label, may catalyzechemical alteration of a substrate compound or composition which isdetectable. A hapten or epitope that is immunospecifically bound by anantibody can also serve as a label. In preferred embodiments, the labelis a fluorescent label. The probe can include one or several labels.When a method is practiced with more than one probe, each differentprobe type is labelled with a different label to allow discriminatingbetween the different probe types.

The term “labeled probe” or “probe associated with a label” refers to aprobe comprising (1) a nucleic acid having a sequence rendering itcapable of hybridizing with a target nucleic acid sequence that isbonded or fused directly or indirectly, or bound with (2) a label.Preferably such hybridization is specific, i.e., it occurs under highstringency conditions.

Probes should have sufficient complementarity to the target nucleic acidsequence of interest so that stable and specific binding occurs betweenthe target nucleic acid sequence and the probe. The degree ofhomology/identity required for stable hybridization varies with thestringency of the hybridization medium and/or wash medium. Preferably,completely homologous probes are employed in the present disclosure, butpersons of skill in the art will readily appreciate that probesexhibiting lesser but sufficient homology can be used in the presentdisclosure (see e.g., Sambrook, J., et al., Molecular Cloning ALaboratory Manual, Cold Spring Harbor Press, (1989)).

Probes may also be generated and chosen by several means including, butnot limited to, mapping by in situ hybridization, somatic cell hybridpanels, or spot blots of sorted chromosomes; chromosomal linkageanalysis; or cloned and isolated from sorted chromosome libraries fromhuman cell lines or somatic cell hybrids with human chromosomes,radiation somatic cell hybrids, microdissection of a chromosome region,or from yeast artificial chromosomes (YACs) or bacterial artificialchromosomes (BAC) identified by PCR primers specific for a uniquechromosome locus or other suitable means like an adjacent YAC or BACclone. Probes may be genomic DNA, eDNA, or RNA cloned in a plasmid,phage, cosmid, YAC, BAC, viral vector, or any other suitable vector.Probes may be cloned or synthesized chemically by conventional methods.When cloned, the isolated probe nucleic acid fragments are typicallyinserted into a vector, such as lambda phage, pBR322, M13, or vectorscontaining the SP6 or T7 promoter and cloned as a library in a bacterialhost (see, e.g., Sambrook, supra).

In one embodiment, the labeled probes are fluorescent and can be used,for example, in a method of fluorescent in situ hybridization (FISH). Insuch embodiments, the probes specifically bind to the target sequence ofa chromosome with a high degree of sequence complementarity. In situhybridization is generally carried out on cells or tissue sections fixedto slides. In situ hybridization may be performed by severalconventional methodologies (see, e.g., Leitch et al., In SituHybridization: A Practical Guide, Oxford BIOS Scientific Publishers,Micropscopy Handbooks v. 27 (1994)). In one in situ procedure,fluorescent dyes (such as fluorescein isothiocyanate (FITC) whichfluoresces green when excited by an Argon ion laser) are used to label anucleic acid sequence probe that is complementary to a target nucleotidesequence in the cell. Each cell containing the target nucleotidesequence will bind the labeled probe producing a fluorescent signal uponexposure, of the cells to a light source of a wavelength appropriate forexcitation of the specific fluorochrome used. FISH analysis can be usedin conjunction with other assays, including without limitationmorphological staining (of serial sections or the same section; see PCTPublication No. WO 00/20641).

Various degrees of hybridization stringency can be employed to allow thebinding/washing of the probe to its complementary target sequence. Asthe hybridization conditions become more stringent, a greater degree ofcomplementarity is required between the probe and target to form andmaintain a stable duplex. Stringency is increased by raisingtemperature, lowering salt concentration, or raising formamideconcentration. Adding dextran sulfate or raising its concentration mayalso increase the effective concentration of labeled probe to increasethe rate of hybridization and ultimate signal intensity. Afterhybridization, slides are washed in a solution generally containingreagents similar to those found in the hybridization solution withwashing time varying from minutes to hours depending on requiredstringency. Longer or more stringent washes typically lower nonspecificbackground but run the risk of decreasing overall sensitivity.

Probes used in the FISH assay may be either RNA or DNA oligonucleotidesor polynucleotides and may contain not only naturally occurringnucleotides but their analogs like digoxygenin dCTP, biotin dcTP7-azaguanosine, azidothymidine, inosine, or uridine. Other useful probesinclude peptide probes and analogues thereof, branched gene DNA,peptidometics, peptide nucleic acid (PNA), and/or antibodies.

Probes are preferably labeled with a fluorophor as the fluorescentlabel. Examples of fluorophores include, but are not limited to, rareearth chelates (europium chelates), Texas Red, rhodamine, fluorescein,dansyl, Lissamine, umbelliferone, phycocrytherin, phycocyanin, orcommercially available fluorophors such Spectrum Orange7 and SpectrumGreen7, and/or derivatives of any one or more of the above. Multipleprobes used in the assay may be labeled with more than onedistinguishable fluorescent or pigment color. These color differencesprovide a means to identify the hybridization positions of specificprobes. Moreover, probes that are not separated spatially can beidentified by a different color light or pigment resulting from mixingtwo other colors (e.g., lightred+green=yellow), pigment (e.g.,blue+yellow=green), or by using a filter set that passes only one colorat a time.

Probes can be labeled directly or indirectly with the fluorescent label,utilizing conventional methodology. Additional probes and colors may beadded to refine and extend this general procedure to include moregenetic abnormalities or serve as internal controls. In one embodiment,FISH probes are directly labeled with the fluorescent label, or iscovalently bonded to a fluorescent label.

Probes can have 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400,500 nucleic acid bases or more in length.

After processing for FISH, the slides may be analyzed by standardtechniques of fluorescence microscopy (see, e.g., Ploem and Tanke,Introduction to Fluorescence Microscopy, 15 Oxford University Press: NewYork (1987)). Briefly, each slide is observed using a microscopeequipped with appropriate excitation filters, dichromic, and barrierfilters. Filters are chosen based on the excitation and emission spectraof the fluorochromes used. Photographs of the slides maybe taken withthe length of time of film exposure depending on the fluorescent labelused, the signal intensity and the filter chosen. For FISH analysis thephysical loci of the cells of interest determined in the morphologicalanalysis are recalled and visually conformed as being the appropriatearea for FISH quantification. In some embodiments, tumor samples treatedwith fluorescent labeled probes are analyzed sample by sample, sectionby section, or on a cell by cell basis.

16p13.3 Gain Detection

As used herein the term “16p13.3” refers to a specific chromosomicregion of chromosome 16 in humans that includes both non-coding andcoding sequences. The chromosomal region 16p13.3 includes, but is notlimited to genes such as: GFER, NTHL1, TSC2, PKD1, RAB26, TRAF7,CASKIN1, MLST8, PGP, E4F1, DNASE1L2, ECI1, RNPS1, ABCA3, ABCA17P, CCNF,NTN3, TBC1D24, ATP6VOC, AMDHD2, CEMP1, PDPK1, KCTDS, PRSS27, and SRRM2.As used in the context of the present disclosure, a 16p13.3 genomic gainincludes a DNA copy number gain of one or more of the chromosomal regionwhich can include the above listed genes. In some embodiments, a 16p13.3genomic gain involves a DNA copy number gain of one or more of PKD1,RAB26, TRAF7, CASKIN1, MLST8, PGP, E4F1, DNASE1L2, ECI1, RNPS1, ABCA3,ABCA17P, CCNF, NTN3, TBC1D24, ATP6VOC, AMDHD2 or CEMP1. In oneembodiment, a 16p13.3 genomic gain involves a DNA copy number gain ofPDPK1. In some embodiments, the gain is more than one and correspond totwo, three, four or more amplifications.

The focal 16p13.3 genomic gain was previously mapped in primary prostatetumors and identified PDPK1 encoding 3-phosphoinositide—dependentprotein kinase-1 (PDK1) as a likely driver of the gain with functionalimpact on prostate cancer cell migration (Choucair et al., 2012).Encoded by PDPK1, PDK1 phosphorylates and activates the AGC kinasemembers regulated by phosphatidylinositol 3-kinase, including AKT. Inaddition to its kinase activity on AKT, it has been shown that PDK1 alsohas an important role in in vitro prostate cancer cell migration(Choucair et al., 2012).

In some embodiments, a 16p13.3 genomic gain comprises a single copy gainof the one or more genes from the 16p13.3 region of chromosome 16. Insome embodiments, a 16p13.3 genomic gain comprises two copy gain, threecopy gain, four copy gain or more of the one or more genes from the16p13.3 region of chromosome 16.

To detect a 16p13.3 genomic gain, methods provided herein comprisecontacting a sample with a first 16p13.3 probe capable of specificallyrecognizing a 16p13.3 chromosome region, wherein the first probe isassociated with a first label; and detecting the signal from the firstlabel of the first probe. The sample is also contacted with a firstreference probe comprising a first reference label to specifically bindto and detect a reference region in chromosome 16. The reference regioncan be any region of chromosome 16 that is not susceptible of beinggained or deleted in the sample. Then, the signal from the first labelis compared relative to the signal from a first reference label. A16p13.3 gain is detected if the incident of signals from the first labelof the first 16p13.3 probe is greater than the incident of signals fromthe first reference label probe. Adjustments can be made to detect a16p13.3 gain, for example when the incident of signals from the firstlabel is at least twice, thrice or more higher than the incidents ofsignal from the first reference label.

In some embodiments, the first 16p13.3 probe hybridizes with a 16p13.3chromosome region of chromosome 16. This hybridization can be observedin the coding as well as in the non-coding sequences of the 16p13.3chromosome regions. In some embodiments, the first 16p13.3 probehybridizes with a chromosome region encoding for one or more of thegenes from the 16p13.3 region of chromosome 16. In one embodiment, thefirst 16p13.3 probe hybridizes with a chromosome region encoding one ormore of the genes: PKD1, RAB26, TRAF7, CASKIN1, GBL, PGP, E4F1,DNASE1L2, DCI, RNPS1, ABCA3, ABCA17, CCNF, NTN3, TBC1D24, KIAA1171,ATP6V0C, AMDHD2, CEMP1, or PDPK1. In some embodiments, the first 16p13.3probe comprises a nucleotide sequence that is complementary to one ormore of the fragments from the 16p13.3 region of chromosome 16, whichcan include, for example, one or more of the genes listed above. In oneembodiment, the first 16p13.3 probe is derived from a BAC clone, suchas, for example, a RP11-20I23 BAC clone.

In the context of the present disclosure, the first 16p13.3 probe can bea variant probe which comprises a nucleic sequence that is complementaryto or hybridizes with a chromosome region encoding a 16p13.3 region. Avariant probe comprises at least one nucleotide difference(substitution, addition, or deletion) when compared the native probe. Avariant includes an allele variant of a gene (dominant or recessive). Inan embodiment, the first 16p13.3 variant probe and has at least 50%,60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to thenative probe and still be able to hybridize specifically to the 16p13.3region. The term “percent identity”, as known in the art, is arelationship between two or more polypeptide sequences, as determined bycomparing the sequences. The level of identity can be determinedconventionally using known computer programs. Identity can be readilycalculated by known methods, including but not limited to thosedescribed in: Computational Molecular Biology (Lesk, A. M., ed.) OxfordUniversity Press, N Y (1988); Biocomputing: Informatics and GenomeProjects (Smith, D. W., ed.) Academic Press, N Y (1993); ComputerAnalysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G.,eds.) Humana Press, N J (1994); Sequence Analysis in Molecular Biology(von Heinje, G., ed.) Academic Press (1987); and Sequence AnalysisPrimer (Gribskov, M. and Devereux, J., eds.) Stockton Press, NY (1991).Preferred methods to determine identity are designed to give the bestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignments of thesequences disclosed herein were performed using the Clustal method ofalignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the defaultparameters (GAP PENALTY=10, GAP LENGTH PEN ALT Y=10). Default parametersfor pairwise alignments using the Clustal method were KTUPLB 1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

The present disclosure also provides first 16p13.3 probe fragmentscomprising a nucleic acid sequence that is complementary to thenucleotide sequences of one or more of the genes from the 16p13.3 regionof chromosome 16 and hybridizes to a chromosome region encoding aportion of the one or more of the genes from the 16p13.3 region. Afragment sequence comprises at least one less nucleotide when comparedto the full-length nucleic acid sequence of the native probe. In anembodiment, the first 16p13.3 probes has a nucleic acid sequence thatexhibits or has at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%,96%, 97%, 98% or 99% identity to the full-length nucleic acid sequenceof the native probe. The fragment sequence can be, for example, atruncation of the full-length sequence of the native probe.Alternatively or in combination, the fragment sequence can be generatedfrom removing one or more internal nucleotides to the native probe. Inan embodiment, the first 16p13.3 probes has a nucleic acid sequence thatis complementary to at least 100, 150, 200, 250, 300, 350, 400, 450, 500or more consecutive nucleotides of the one or more of the native probe.

The first reference probe is specific for a reference location onchromosome 16 and, in some embodiments, is capable of specificallyrecognizing a 16qh centromeric region. In one embodiment, the firstreference probe is derived from a pHuR-195 BAC clone, variants thereof,fragments thereof or complements thereof.

In the context of the present disclosure, the first reference probecomprises a variant nucleic sequence that is complementary to orhybridizes with a 16qh centromeric region including variants of one ormore of the genes from the 16qh centromeric region of chromosome 16. Avariant probe comprises at least one nucleotide difference(substitution, addition, or deletion) when compared to the native probe.A variant can include, for example, an allele variant of a gene in thechromosomal region (dominant or recessive). In an embodiment, the firstreference variant probe hybridizes with a 16qh centromeric region ofchromosome 16 and has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% identity to the nucleic acid sequence of the nativeprobe. The term “percent identity”, as known in the art, is arelationship between two or more polypeptide sequences, as determined bycomparing the sequences. The level of identity can be determinedconventionally using known computer programs. Identity can be readilycalculated by known methods, including but not limited to thosedescribed in: Computational Molecular Biology (Lesk, A. M., ed.) OxfordUniversity Press, N Y (1988); Biocomputing: Informatics and GenomeProjects (Smith, D. W., ed.) Academic Press, N Y (1993); ComputerAnalysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G.,eds.) Humana Press, N J (1994); Sequence Analysis in Molecular Biology(von Heinje, G., ed.) Academic Press (1987); and Sequence AnalysisPrimer (Gribskov, M. and Devereux, J., eds.) Stockton Press, NY (1991).Preferred methods to determine identity are designed to give the bestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignments of thesequences disclosed herein were performed using the Clustal method ofalignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the defaultparameters (GAP PENALTY=10, GAP LENGTH PEN ALT Y=10). Default parametersfor pairwise alignments using the Clustal method were KTUPLB 1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

The present disclosure also provides a first reference probe fragmentcomprising a nucleic acid sequence that is complementary to the 16qhcentromeric region of chromosome 16 and hybridizes to a chromosomeregion encoding a portion of the one or more of the genes from the 16qhcentromeric region. A fragment sequence comprises at least one lessnucleotide when compared to the full-length nucleic acid sequence of thenative probe or variant thereof. In an embodiment, the first referencefragment probe has a nucleic acid sequence that exhibits or has at least30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identity to the full-length nucleic acid sequence of the native probe.The fragment sequence can be, for example, a truncation of thefull-length sequence of the native probe. Alternatively or incombination, the fragment sequence can be generated from removing one ormore internal nucleotides form the native probe. In an embodiment, thefirst reference fragment probe has a nucleic acid sequence that iscomplementary to at least 100, 150, 200, 250, 300, 350, 400, 450, 500 ormore consecutive nucleotides of the one or more of the native probe.

PTEN Deletion Detection

The phosphatidylinositol 3-kinase (PI3K)/AKT signal transduction pathwaycontributes to cancer growth and survival, and is activated in a broadrange of human malignancies including prostate cancer. The phosphataseand tensin homologue deleted on chromosome 10 (PTEN) is a tumorsuppressor gene on 10q23.3 locus that acts by negatively regulating thePI3K/AKT pathway. PTEN genomic deletion has been detected in humantissues representing all stages of prostate cancer development andprogression including High Grade Prostatic Intraepithelial Neoplasia(HGPIN), primary PCa and at higher frequency in metastatic prostatecancer and castrate resistant prostate cancer.

To detect a PTEN genomic deletion, methods are provided comprisingcontacting a sample with a second (PTEN) probe capable of specificallyrecognizing a 10q23.3 (PTEN) chromosome region, wherein the second probeis associated with a second label; and detecting the signal from thesecond label. The sample is also contacted with a second reference probecomprising a second reference probe comprising a second reference labelto specifically bind to and detect a reference region in chromosome 10.The reference region can be any region of chromosome 10 that is notsusceptible of being gained or deleted in the sample. Then, the signalof the second label is compared to the signal of the second referencelabel. A PTEN deletion is detected if the incidents of signals from thesecond label of the second probe is less than the incidents of signalfrom the second reference label from the second reference probe.Adjustments can be made to detect a PTEN deletion, for example, when theincident of signals from the first label is at least twice, thrice ormore lower than the incidents of signal from the first reference label.

In some embodiments, the second PTEN probe hybridizes with a 10q23.3region of chromosome 10. In some embodiments, the second PTEN probehybridizes with a chromosome region encoding the PTEN gene. In someembodiments, the second PTEN probe comprises a nucleotide sequence thatis complementary to the PTEN gene. In one embodiment, the second PTENprobe is derived from a trans a BAC clone, such as, for example, aCTD-2557P6 BAC clone, variants thereof, fragments thereof, orcomplements thereof.

In some embodiments, a sample comprises cells having an homozygous PTENdeletion where both copies of PTEN are deleted. In other embodiments, asample comprises cells having an heterozygous PTEN deletion where onecopy of PTEN is deleted.

In the context of the present disclosure, the second PTEN probe can be avariant which comprises a nucleic sequence that is complementary to orhybridizes the 10q23.3 region. A variant of a probe comprises at leastone nucleotide difference (substitution, addition, or deletion) whencompared to the native probe. A variant can include an allele variant ofa gene in the 10q23.3 region (dominant or recessive). In an embodiment,the second PTEN variant probe hybridizes with the 10q23.3 region ofchromosome 10 and has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% identity to the nucleic acid sequence of the nativeprobe. The term “percent identity”, as known in the art, is arelationship between two or more polypeptide sequences, as determined bycomparing the sequences. The level of identity can be determinedconventionally using known computer programs. Identity can be readilycalculated by known methods, including but not limited to thosedescribed in: Computational Molecular Biology (Lesk, A. M., ed.) OxfordUniversity Press, N Y (1988); Biocomputing: Informatics and GenomeProjects (Smith, D. W., ed.) Academic Press, N Y (1993); ComputerAnalysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G.,eds.) Humana Press, N J (1994); Sequence Analysis in Molecular Biology(von Heinje, G., ed.) Academic Press (1987); and Sequence AnalysisPrimer (Gribskov, M. and Devereux, J., eds.) Stockton Press, NY (1991).Preferred methods to determine identity are designed to give the bestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignments of thesequences disclosed herein were performed using the Clustal method ofalignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the defaultparameters (GAP PENALTY=10, GAP LENGTH PEN ALT Y=10). Default parametersfor pairwise alignments using the Clustal method were KTUPLB 1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

The present disclosure also provides second PTEN probe fragmentscomprising a nucleic acid sequence that is complementary the 10q23.3region of chromosome 10 and hybridizes to a chromosome region encoding aportion of the PTEN gene from the 10q23.3 region. A fragment sequencecomprises at least one less nucleotide when compared to the full-lengthnucleic acid sequence of the native probe. In an embodiment, the secondPTEN variant probe has a nucleic acid sequence that exhibits or has atleast 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identity to the full-length nucleic acid sequence of the native probe.The fragment sequence can be, for example, a truncation of thefull-length sequence of the native probe. Alternatively or incombination, the fragment sequence can be generated from removing one ormore internal nucleotides from the native probe. In an embodiment, asecond PTEN probe has a nucleic acid sequence that is complementary toat least 100, 150, 200, 250, 300, 350, 400, 450, 500 or more consecutiveamino acids of the native probe or the variant second probe.

The second reference probe is specific for a reference location onchromosome 10 and, in some embodiments, is capable of specificallyrecognizing a 10p11.1-q11.1 centromeric region. In one embodiment, thesecond reference chromosome 10 probe is derived from a CEP10 probe, avariant thereof, a fragment thereof or a complement thereof.

In the context of the present disclosure, the second reference probecomprises variant nucleic sequence that is complementary to orhybridizes with a 10p11.1-q11.1 centromeric region. A variant of a probecomprises at least one nucleotide difference (substitution, addition, ordeletion) when compared to the native probe. A variant can include anallele variant of a gene present in the 10p11.1-q11.1 centromeric region(dominant or recessive). In an embodiment, the second reference variantprobe hybridizes with the 10p11.1-q11.1 centromeric region of chromosome10 and has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% identity to the nucleic acid sequence of the native probe. The term“percent identity”, as known in the art, is a relationship between twoor more polypeptide sequences, as determined by comparing the sequences.The level of identity can be determined conventionally using knowncomputer programs. Identity can be readily calculated by known methods,including but not limited to those described in: Computational MolecularBiology (Lesk, A. M., ed.) Oxford University Press, N Y (1988);Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.)Academic Press, N Y (1993); Computer Analysis of Sequence Data, Part I(Griffin, A. M., and Griffin, H. G., eds.) Humana Press, N J (1994);Sequence Analysis in Molecular Biology (von Heinje, G., ed.) AcademicPress (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux,J., eds.) Stockton Press, NY (1991). Preferred methods to determineidentity are designed to give the best match between the sequencestested. Methods to determine identity and similarity are codified inpublicly available computer programs. Sequence alignments and percentidentity calculations may be performed using the Megalign program of theLASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).Multiple alignments of the sequences disclosed herein were performedusing the Clustal method of alignment (Higgins and Sharp (1989) CABIOS.5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALT Y=10). Default parameters for pairwise alignments using the Clustalmethod were KTUPLB 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

The present disclosure also provides second reference probe fragmentscomprising a nucleic acid sequence that is complementary to the10p11.1-q11.1 centromeric region of chromosome 10 and hybridizes to the10p11.1-q11.1 centromeric region. A fragment sequence comprises at leastone less nucleotide when compared to the full-length nucleic acidsequence of the native probe. In an embodiment, the second referenceprobe fragment has a nucleic acid sequence that exhibits or has at least30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identity to the full-length nucleic acid sequence of the native probeand hybridizes to the 10p11.1-q11.1 centromeric region of chromosome 10,or complements thereof. The fragment sequence can be, for example, atruncation of the full-length sequence of the native probe or variant.Alternatively or in combination, the fragment sequence can be generatedfrom removing one or more internal nucleotides from the native probe. Inan embodiment, a second reference probe fragment has a nucleic acidsequence that is complementary to at least 100, 150, 200, 250, 300, 350,400, 450, 500 or more consecutive amino acids of the native probe.

Identifying and Classifying Cancer Risk

In some embodiments, methods of prognosing a subject with prostatecancer involve classifying the cancer risk of the subject based on thepresence or absence of 16p13.3 gain. As shown herein, the presence of a16p13.3 gain is associated with the presence of prostate cancer andgenerally a poor prognosis. In one embodiment, classifying the cancerrisk of the subject is based on the presence or absence of 16p13.3 gainin combination with a PTEN deletion. As shown herein, the presence of a16p13.3 gain and a PTEN deletion is associated with the presence ofprostate cancer and generally a poor prognosis. The detection of the16p13.3 gain and of the PTEN deletion can be done, for example ondifferent samples from the subject (for example different tissuesections of a sample) or can be multiplexed on a single sample from thesubject.

In some embodiments, methods of determining the presence or absence ofaggressive prostate cancer in a subject involve characterizing the tumorsample based on the presence or absence of 16p13.3 gain, and thepresence of 16p13.3 gain is associated with aggressive prostate cancer.In one embodiment, characterizing the tumor sample is based on thepresence or absence of 16p13.3 gain and PTEN deletion, and the presenceof 16p13.3 gain and PTEN deletion is associated with aggressive prostatecancer.

In some embodiments, methods of predicting recurrence-free and/ormetastasis-free survival in a subject having prostate cancer involvecharacterizing if the subject has 16p13.3 gain, and having 16p13.3 gainis associated with a reduction of recurrence-free and/or metastases-freesurvival. In one embodiment, a method of predicting recurrence-freeand/or metastasis-free survival in a subject having prostate cancerinvolves characterizing if the subject has 16p13.3 gain and PTENdeletion, where having 16p13.3 gain and PTEN deletion is associated witha reduction in the recurrence-free and/or metastases-free survival.

The method of the present disclosure can include classifying thesubjects into risk groups based on clinical features or Gleason grade orCAPRA-S scores. In some embodiments, subjects are classified into low tointermediate risk groups. In some embodiments, the subjects areclassified into high risk groups. The present disclosure also providescharactering the subject as being associated with higher cancer risk, ora higher cancer risk/poor prognosis relative to the assigned riskedgroup based on clinical features, if the subject is characterized ashaving 16p13.3 gain; if the subject is characterized as having PTENdeletion; or if the subject is characterized as having 16p13.3 gain andPTEN deletion.

In some embodiments, a higher cancer risk is associated with a reductionin recurrence free survival or metastasis free survival. In someembodiments, the cancer risk is a risk classification of the cancer andthe higher cancer risk is associated with the association of the subjectwith a more aggressive class of the cancer. In one embodiment, methodsprovided herein allows for further classification into risk sub-groups.In one embodiment, methods provided herein allows for furtherclassification of low to intermediate risk groups into furthersub-groups.

The methods described herein can help in characterizing the subject asbeing associated with a poor prognosis if the subject has the 16p13.3gain and optionally the PTEN deletion or both chromosomal anomalies.This poor prognostic can be associated with a reduction in survival(including recurrence-free and metastasis-free survival) or anaffliction by a more aggressive cancer.

Prostate Cancer Treatment

The present disclosure also provides treating the subject with adjuvantor neoadjuvant therapy if the subject is characterized as beingassociated with higher cancer risk. The term “adjuvant therapy” refersto a therapy that is given in addition to the primary or initial therapyto maximize its effectiveness. The term “neoadjuvant therapy” refers tothe administration of therapeutic agents before a main treatment.

Examples of primary or main treatment for prostate cancer include butare not limited to surgical therapy, such as, transurethral resection ofthe prostate (TURP), prostatectomy, transurethral incision of theprostate (TUIP), transurethral vaporization of the prostate (TUVP),photoselective vaporization of the prostate (PVP), prostatic urethrallift (PUL), transurethral microwave therapy (TUMT), water vapor thermaltherapy, transurethral needle ablation (TUNA), laser enucleation,prostate artery embolization (PAE), cryosurgery; radiation therapy, suchas, external beam radiation therapy, bracytherapy; hormonal therapy,such as, androgen deprivation therapy, antiandrogens, androgen synthesisinhibitors, GnRH antagonists, abiraterone acetate; chemotherapy withcytotoxic agents, or immunotherapy.

In some embodiments, subjects with prostate cancer are treated withradical prostatectomy or radiation therapy as primary therapy.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. I¹³¹,I¹²⁵, Y⁹⁰, and Re¹⁸⁶), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as docetaxel, thiotepa and cyclosphosphamide(CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; nitrogenmustards such as chlorambucil, chlomaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, carzinophilin,chromomycins, dactinomycin, daunorubicin, 6-diazo-5-oxo-L-norleucine,doxorubicin, epirubicin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, streptonigrin,streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folicacid analogues such as denopterin, methotrexate, pteropterin,trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,thiamiprine, thioguanine; pyrimidine analogs such as ancitabine,azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,doxifluridine, enocitabine, floxuridine, 5-FU; androgens such ascalusterone, dromostanolone propionate, epitiostanol, mepitiostane,testolactone; anti-adrenals 25 such as aminoglutethimide, mitotane,trilostane; folic acid replenisher such as frolinic acid; aceglatone;aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil;bisantrene; edatraxate; defofamine; demecolcine; diaziquone;elfomithine; elliptinium acetate; etoglucid; gallium nitrate;hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol;nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid;2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofiran;spirogermanium; tenuazonic acid; triaziquone; 2, 2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytos:ine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddoxetaxel (Taxotere™, Rhone-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;carminomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomeraseinhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid;esperamicins; capecitabine; and pharmaceutically acceptable salts, acidsor derivatives of any of the above. Also included in this definition arehormonal agents that act to regulate or inhibit hormone action on tumorssuch as anti-estrogens including for example tamoxifen, raloxifene,aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene,keoxifene, LY117018, onapristone; and anti-androgens such as flutanlideand nilutamide; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

Examples of antiandrogens are flutamide, nilutamide, bicalutamide,enzalutamide, apalutamide, or cyproterone acetate. Examples of androgensynthesis inhibitors are ketoconazole, anioglutethimide or abiraterone.Examples of GnRH antagonists are abarelix or degarelix.

In some embodiments, an adjuvant therapy comprises one or more ofsurgical therapy, radiation therapy, hormonal therapy, chemotherapy, orimmunotherapy. In some embodiments, neoadjuvant comprises one or more ofsurgical therapy, radiation therapy, hormonal therapy, chemotherapyimmunotherapy. In one embodiment, the primary therapy is surgicaltherapy and/or radiation therapy, and the adjuvant and/or neoadjuvanttherapy is one or more of hormonal therapy, chemotherapy, orimmunotherapy. In one embodiment, the primary therapy is a firsthormonal therapy, chemotherapy, and/or immunotherapy, and the adjuvantand/or neoadjuvant therapy is a second hormonal therapy, chemotherapy,and/or immunotherapy.

In the context of the present disclosure, methods of treating a subjecthaving prostate cancer with an adjuvant or neoadjuvant therapy isprovided. The method involves performing the methods described herein onthe tumor sample to determine if the subject has a 16p13.3 gain, andcharacterizing the cancer risk of the subject. If the subject has highercancer risk, then the subject is administered adjuvant or neoadjuvanttherapy. In one embodiment, further microscopy assay is performed todetermine if the subject has a PTEN deletion. In some embodiments, themethods include performing a microscopy assay is a fluorescentmicroscopy assay. In one embodiment, the microscopy assay is FISH.

In some embodiments, characterizing the patient as having higher cancerrisk or poor prognosis comprises characterizing the subject as having16p13.3 gain; characterizing the subject as having PTEN deletion; orcharacterizing the subject as having 16p13.3 gain and PTEN deletion.

In one embodiment, a subject characterized has having higher cancer riskis treated with abiraterone plus prednisone, enzalutamide or docetaxel.In one embodiment, a subject characterized has having higher cancer riskis treated with ketoconazole plus steroid, mitoxantrone or radionuclidetherapy. In one embodiment, a subject characterized has having highercancer risk is treated with radium²²³. In one embodiment, a subjectcharacterized has having higher cancer risk is treated with docetaxel ormitoxantrone chemotherapy. In one embodiment, a subject characterizedhas having higher cancer risk is treated with abiraterone plusprednisone, cabazitaxel or enzalutamide. In one embodiment, a subjectcharacterized has having higher cancer risk is treated with docetaxel.In one embodiment, a subject characterized has having higher cancer riskis treated with abiraterone plus prednisone, enzalutamide, ketoconazoleplus steroid or radionuclide therapy.

In one embodiment, a subject characterized has having higher cancer riskis treated neoadjuvant therapy or preventative therapy comprisingdenosumab or zoledronic acid.

Kits for Assessing Cancer Risk

The present disclosure also provides kits for assessing cancer risk ofsubject, such as subjects having prostate cancer. The kit includes afirst probe capable of specifically recognizing a 16p13.3 chromosomeregion and a first reference probe capable of specifically recognizing areference region of chromosome 16. In some embodiments, the first probehybridizes with a region of chromosome 16 encoding one or more genesencoded in the 16p13.3 chromosome region. In one embodiment, the firstprobe hybridizes with one or more of PKD1, RAB26, TRAF7, CASKIN1, MLST8,PGP, E4F1, DNASE1L2, ECI1, RNPS1, ABCA3, ABCA17A, CCNF, NTN3, TBC1D24,ATP6VOC, AMDHD2, CEMP1, PDPK1, variants thereof, fragments thereof, orcomplements thereof. In one embodiment, the first probe is derived froma RP11-20I23 BAC clone, variants thereof, fragments thereof, orcomplements thereof. In some embodiments, the first reference probehybridizes with a 16qh centromeric region.

In some embodiments, the kit further includes a second probe capable ofspecifically recognizing a 10q23.3 (PTEN) chromosome region and a secondreference probe capable of specifically recognizing a reference regionof chromosome 10. In some embodiments, the second probe hybridizes witha region of chromosome 10 encoding the PTEN gene, variants thereof,fragments thereof, or complements thereof. while the second referenceprobe hybridizes with a 10p11.1-q11.1 centromeric region. In oneembodiment, the second probe is derived from a CTD-2557P6 BAC clone,variants thereof, fragments thereof, or complements thereof.

In some embodiments, the probes are fluorescent labeled probes, such asFISH probes. The kit may also include other reagents needed forperforming fluorescent microscopy (i.e. FISH), such as further imagingreagents, dyes, contrasting medium, and/or buffers.

The present invention will be more readily understood by referring tothe following examples which are given to illustrate the inventionrather than to limit its scope.

Example I—Materials and Methods for 16P13.3 Fish Analysis

Study population and tissue microarray. This study was conducted withthe written informed consent of the participants and approval from theResearch Ethics Board of McGill University Health Centre (Quebec,Canada, BDM-10-115). This biomarker study was done in accordance withREMARK guidelines (McShane et al., 2005). FFPE RP tissue specimens(n=304) collected between 1993 and 2008 at the McGill University HealthCentre were represented on a tissue microarray (TMA) by duplicate 1-mmcores taken from the dominant tumor nodule. The clinical correlates wereretrieved from the medical chart and pathologic data were obtained byre-review of all RP cases by a single dedicated genitourinarypathologist. The recent 2014 International Society of UrologicalPathology (ISUP) criteria were used for assigning the final grade(Epstein et al, 2016). The clinicopathologic characteristics of thestudy subjects are summarized in Table 1. Briefly, the mean preoperativeserum PSA level for the cohort was 8.60 (±8.21), and the distribution ofGS 6, 7, and was 20.4%, 70.4%, and 9.2%, respectively. Sixty-fourpercent of patients belonged to pT2-stage, while 36% were at stage pT3.Cases for which the serum PSA did not fall to undetectable levelspostsurgery were considered as surgical failure and were not included inbiochemical recurrence analyses (n=14). Patients who receivedneoadjuvant hormone therapy (n=5) and cases with missing serum PSA datapost-surgery were also excluded from BCR analyses (n=15). Biochemicalrecurrence (BCR) was defined by serum PSA elevation of >0.2 ng/mL afterRP (29%), and the recurrence-free interval was defined as the timebetween the date of surgery and the date of first PSA increase above 0.2ng/mL. Patients with no BCR were censored at the last follow-up datewith a PSA measurement. The median follow-up for the cohort was 118months (1-253 months, min-max). The metastatic status was confirmed byimaging in patients with clinical signs or symptoms (n=16). Themetastasis-free interval was defined as the time between the date ofsurgery and the date of first metastasis detection. Patients with nosigns/symptoms of metastasis were censored at the last follow-up/PSAdate. The Cancer of the Prostate Risk Assessment Post-Surgical (CAPRA-S)score was calculated on the basis of the status of six clinicopathologicvariables [preoperative PSA, GS, surgical margin (SM), extracapsularextension, seminal vesicle invasion, lymph node invasion], and eachpatient was assigned to one of the three risk groups: low (0-2),intermediate (3-5), and high (6) according to Cooperberg and colleagues(Cooperberg et al., 2011). Of note, patients who did not undergo a lymphnode dissection were deemed to have negative lymph nodes for CAPRA-Sscore calculation as described previously (Punnen et al., 2014). Theprostate cancer DNA CNAs profiling data reported by Taylor andcolleagues (Taylor et al., 2010) was used for validation, and theclinical correlates were derived directly from the MSKCC Prostate CancerGenomics Data Portal.

TABLE 1 Clinicopathologic features of radical prostatectomy casesrepresented on the tissue microarray. Clinicopathologic variablesCategory n (%) Total number of cases N 304 Age (years) Median  61Min-max 43-73 Preoperative PSA (ng/mL) n^(a) 298 Mean (±SD) 8.60 (±8.21)PSA < 10 232 (78%) PSA ≥ 10 66 (22%) GS at surgery GS 6 62 (20.4%) GS 7214 (70.4%) GS ≥ 8 28 (9.2%) Pathologic stage (T-stage) pT2 195 (64%)pT3A 87 (29%) PT3B 22 (7%) Surgical margin status Positive 91 (30%)Follow-up (months) n^(a) 270 Median (min-max) 118 (1-253) BCR n^(a) 270Positive 78 (29%) Metastases Positive 16/293^(a) (5.4%) ^(a)Values notavailable for all the 304 cases (n noted for each variable).

Fluorescence in situ hybridization (FISH). Dual-color FISH was performedon TMA sections using as probes, a 16p13.3 specific BAC clone RP11-20I23(BACPAC Resources Center) and the recombinant DNA clone pHuR-195 (ATCC),mapping to the 16qh centromeric region (Moyzis et al., 1987). RP11-20I23and pHuR-195 DNA were labeled with Spectrum OrangedUTP and SpectrumGreen-dUTP (Enzo Life Sciences) respectively, using Nick TranslationReaction Kit (Abbott Molecular) and were used to perform FISH on 5-mmTMAsections as described previously (Choucair 2012).

FISH data analysis. To evaluate the 16p13.3 copy number status,fluorescent signals were counted in 100 non-overlapping interphasenuclei for each case (as identified on corresponding H&E) counterstainedwith ProLong Gold antifade reagent with DAPI (Life Technologies), todelineate nuclei. The 16p13.3 gain was defined as present at a thresholdof ≥15% of tumor nuclei containing three or more 16p13.3 locus signalsand two pHuR-195 signals, as previously reported (Choucair 2012). Imageswere acquired with an Olympus IX-81 inverted microscope at X96magnification, using Image-Pro Plus 7.0 software (Media Cybernetics).

Statistical analysis. Associations between the 16p13.3 gain andclinicopathologic variables were evaluated by Fisher exact test fordichotomous variables and unpaired t test for continuous variables.Kaplan-Meier method and the log-rank test were used to generate andcompare recurrence-free survival and metastasis-free survival curves,respectively. Cox regression analyses and the Wald test were used toevaluate univariate and multivariate HRs. The C-index was calculated asdescribed by Harrell and colleagues (Harrell et al., 1996). Analyseswere performed using SPSS, WinStat, and R (Version 3.3.2).

Example II—Results of 16P13.3 Fish Analysis

Association of 16p13.3 gain with adverse clinicopathologic features inRP. Dual-color FISH was used to assess CNA at chromosome 16p13.3 on 304RP specimens represented on a TMA. The clinicopathologic characteristicsof the study subjects are summarized in Table 1. A total of 267 primarytumors were scorable by FISH, among which 113 (42%) harbored significant16p13.3 genomic gain (FIG. 1A to 1D). The 16p13.3 gain was significantlyassociated with clinicopathologic features of aggressive prostate cancer(Table 2) including high preoperative serum PSA levels (P=0.03), GS(P<0.0001), advanced pT-stage (P<0.0001), and positive SMs (P=0.009).The level of gain was restricted to single copy gain in most specimens,whereas 20 cases with 16p13.3 gain exhibited more than three copies ofthe locus in at least 10% of their nuclei. As compared with theorgan-confined stage-T2 tumors, the advanced stage-T3 tumors harboredsignificantly higher percentage of nuclei with more than three copies(Mann-Whitney U test, P=0.01).

TABLE 2 Association of 16p13.3 gain with clinicopathologic features ofaggressive prostate cancer. Total Cases 16p13.3 Status Variables_McGillCohort n (%) No gain Gain P-value 16p13.3 Status 267 154 (58%) 113 (42%)Age (years) Mean (±SD) 61.8 (±5.8) 61.0 (±6.12) 60.7 (±5.64) 0.68^(†)Preoperative PSA (Mean/±SD)  261* 7.6 (±6.88) 9.61 (±8.28) 0.03^(†)Gleason Score 267 <0.0001^(‡) GS = 6  59 49 (83%) 10 (7%) GS = 7 185 98(53%) 87 (47%) GS ≥ 8  23 7 (30%) 16 (70%) Pathological T-Stage 267<0.0001^(‡) pT2 176 118 (67%) 58 (33%) pT3A  72 31 (43%) 41 (57%) pT3B 19 05 (26%) 14 (74%) Surgical Margins 267 0.009 Negative 189 119 (63%)70 (37%) Positive  78 35 (45%) 43 (55%) ^(†)unpaired t-test^(‡)two-sided Fisher's exact test *Values not available for all the 267cases that could be assessed by FISH (n noted for each variable)

Prognostic significance of 16p13.3 genomic gain in prostate cancer. Theimpact of the 16p13.3 genomic gain was assessed on clinical outcomeusing BCR as a surrogate primary endpoint following RP. Of the 238 casesfor which both FISH and complete PSA follow-up data were available(median follow up=117 months), 65 (26%) experienced BCR. TheKaplan-Meier analysis revealed that the 16p13.3 gain was significantlyassociated with shorter BCR-free survival following RP (logrankP=0.0005; FIG. 2A). The 16p13.3 gain was then assessed in terms of itsability to stratify patients with low-intermediate risk, representing amajority of patients encountered in clinical practice. It was observedthat 16p13.3 gain status could further risk stratify patients for BCRwhen considering the subgroup of patients with either preoperative PSA10or GS7 (log-rank P=0.02 and P=0.006, respectively; FIGS. 2B and 2C), butnot significantly in the pT-stage subgroup (log-rank P=0.24; FIG. 2D).

Although the main endpoint of this study was BCR, secondary endpointswere also evaluated, such as bone or soft tissue metastases. The 16p13.3gain status was significantly associated with an increased risk ofmetastases (log-rank P=0.03; FIG. 2E), supporting its potential utilityas a marker of prostate cancer progression.

Improved risk stratification of existing prognostic tools when combinedwith 16p13.3 gain. The HR for the 16p13.3 gain (HR=2.30; 95% CI,1.42-3.84; P=0.001) was estimated by univariate Cox regression analysisalong with preoperative PSA levels, GS, pT-stage, and SM, which werealso significantly associated with BCR (Table 3). In multivariateanalysis, the 16p13.3 gain status remained a significant predictor ofBCR after adjusting for the above standard clinicopathologic variables(Table 3). These observations were validated in an independent datasetfrom Taylor and colleagues (Taylor at al., 2010), who reported DNA CNAsby array-CGH analyses of 194 prostate cancer cases with clinicalfollow-up (Table 4).

TABLE 3 Univariate and multivariate Cox proportional hazards analysisfor 16p13.3 gain adjusting for standard clinicopathologic parameters.McGill Cohort Univariate Analysis Multivariate Analysis Variable HR 95%CI P-value* HR 95% CI P-value* Preoperative PSA^(†) 1.07 1.04-1.10<0.0001 1.05 1.02-1.08 <0.0001 Gleason Score 5.69  3.01-10.76 <0.00013.06 1.47-6.38 0.003 (≥8 vs ≤GS7) pT-stage (T3 vs T2) 3.46 2.10-5.69<0.0001 1.96 1.10-3.48 0.02 16p13.3 Status 2.30 1.40-3.78 0.001 1.711.01-2.89 0.04 (Gain vs No Gain) Surgical Margin 2.14 1.30-3.51 0.0031.53 0.88-2.64 0.12 (Positive vs Negative) ^(†)Analyzed as continuousvariable; HR: Hazard ratio; CI: Confidence Interval *Wald test

TABLE 4 Univariate and Multivariate Cox Proportional Hazard Analysis for16p13.3 Gain in Taylor et al. Validation Dataset Taylor et al. CohortUnivariate Analysis Multivariate Analysis Variable HR 95% CI P-value* HR95% CI P-value* Preoperative PSA^(†) 1.005 1.002-1.007 <0.0001 1.0071.004-1.01  <0.0001 Gleason Score 7.60  4.42-13.07 <0.0001 6.44 3.57-11.61 <0.0001 (≥8 vs ≤GS7) pT-stage (T3 vs T2) 3.42 1.95-5.99<0.0001 2.40 1.24-4.64 0.009 16p13.3 Status 4.95  2.09-11.74 <0.00014.50  1.68-12.04 0.003 (Gain vs No Gain) Surgical Margin 1.82 1.07-3.100.02 1.21 0.64-2.30 0.54 (Positive vs Negative) ^(†)Analyzed ascontinuous variable; HR: Hazard ratio; CI: Confidence Interval *Waldtest

It was further evaluated whether combining 16p13.3 gain status withpreoperative PSA, GS, and pT-stage, respectively, would improve patientstratification. The combination of the 16p13.3 gain status with each ofthese standard clinicopathologic variables segregated prostate cancercases into three prognostic subgroups (log-rank P<0.0001, respectively;FIG. 3A to 3C) stratified by the number of positive markers: (i) worstprognostic group characterized by presence of both markers; (ii)intermediate prognostic group with either of the two positive markers;and (iii) favorable prognostic group, with both markers absent.Furthermore, the BCR risk stratification significantly improved when allthe 4 variables were considered to group the patients (log-rank test,P<0.0001; FIG. 3D).

It was then explored whether the 16p13.3 gain status could providefurther prognostic information to the CAPRA-S score, a recentlydeveloped and validated clinicopathologic tool to predict the risk ofrecurrence post RP (Cooperberg et al., 2011). In multivariate analysis,the 16p13.3 gain status was a significant predictor of BCR along theCAPRA-S score risk groups (Table 5). As expected, the three risk groupsdefined by the CAPRA-S score were associated with distinct BCR-freesurvival probabilities (FIG. 4A). It was then assessed whether the16p13.3 gain status could further stratify each of these risk groups.Although the 16p13.3 gain status did not further stratify the low-riskgroup, cases in the intermediate-risk group harboring the gain presenteda similar risk of BCR as the high CAPRA-S risk group without thisgenomic alteration, while those belonging to the high CAPRA-S risk groupwith the gain had the worst outcome (FIG. 4B). By merging groups withthe overlapping risk of BCR, four risk groups were delineated (FIG. 4C).The addition of 16p13.3 gain status to the CAPRA-S score further led tothe increase of the C-index as compared with the CAPRA-S score alone(0.78 vs. 0.77). Similarly, in Taylor and colleagues validation dataset,the gain status with CAPRA-S score also identified cases with very highrisk of recurrence with a C-index of 0.73 as compared with 0.72 for theCAPRA-S alone (FIGS. 5A and 5B).

TABLE 5 Univariate and multivariate Cox proportional hazards analysisfor CAPRA-S score risk groups and 16p13.3 gain. McGill Cohort UnivariateAnalysis Multivariate Analysis Variable HR 95% CI P-value* HR 95% CIP-value* CAPRA-S Risk Low (0-2) Ref. <0.0001 <0.0001 Intermediate (3-5)3.13 1.66-5.91 0.0004 2.91 1.54-5.51 0.001 High (≥6) 8.94  4.49-17.79<0.0001 7.82  3.89-15.73 <0.0001 16p13.3 Status (Gain vs No Gain) 2.301.40-3.78 0.001 1.81 1.09-3.00 0.02 HR: Hazard ratio; CI: ConfidenceInterval *Wald test

Example III—Role of 16P13.3 Gain in Prostate Cancer Progression and asPrognostic Biomarker

The detection of 16p13.3 gain in primary prostate cancer specimens asshown above in Examples I and II was in coherence with previouslypublished report, where Choucair and colleagues detected 16p13.3 gain byFISH in 20% of the 46 RP specimens assessed (Choucair et al., 2012). Thedifference in the proportion of cases harboring the gain between theprevious study (20%) and the present study (42%) might be attributed tothe small sample size of the earlier study (n=46) or reflected realbiological differences between these two independent patient sets. Themajority of cases with 16p13.3 gain harbored a single extra copy. Singlecopy gains of loci or entire chromosome are known recurrent events inother types of cancer that effectively contribute to tumor phenotypes(Abel et al., 1999; An et al., 2014; Bown et al., 1954; Towle et al.,2014). A few cases harbored more than a single copy gain, and they weremore prevalent in the pT3-stage tumors.

The 16p13.3 genomic gain was associated with clinicopathologic featuresof aggressive prostate cancer, such as high GS and preoperative PSAlevels. Patients harboring 16p13.3 gain were more than twice as likelyto experience BCR following RP than those without the gain. Combiningthe 16p13.3 gain status with individual clinicopathologic markerssignificantly improved BCR risk stratification in this study, whereinthe incremental number of positive variables was associated with ahigher risk of BCR, which reached its maximum for patients with fouradverse factors, including the 16p13.3 gain. This improvedstratification was observed in patients with intermediate and high riskof disease progression based on their CAPRA-S score. These results arein line with previous reports showing that genomic markers can furtherstratify subsets of patients classified by the CAPRA-S score (Cooperberget al., 2015; Lennartz et al., 2016). The fact that the CAPRA-S score isalready a very strong multiparameter predictor of BCR and that thelow-risk group was not further stratified by the 16p13.3 gain mayexplain the limited increase of the C-index observed by the addition ofthis single variable to the model. Further validation studies arewarranted on larger cohorts to further evaluate the added prognosticvalue of the 16p13.3 gain to the clinicopathologic predictors.Nevertheless, the addition of the 16p13.3 gain status to CAPRA-Sidentified a subset of patients at very high risk of recurrence, who maybenefit from adjuvant treatments after RP.

Although very few metastatic events were observed in the McGill cohort(approximately 5%), the 16p13.3 gain was also predictive of theincreased risk of developing distant metastases following RP. Recently,using the whole-genome sequencing approach, Beltran and colleaguesdetected the 16p13.3 gain in 52% of the metastatic CRPC tumor samples(Beltran et al., 2016). These results are in line with previous studiesdetecting gain in about 50% prostate cancer lymph node metastases, anoverrepresentation as compared with unpaired primary tumors (Lapointe etal., 2007; Choucair et al., 2012). These observations further support arole of 16p13.3 gain in cancer progression and warrant future studies inthe context advanced diseases and response to therapies includingandrogen ablation.

The association of 16p13.3 gain with features of aggressive tumors wasin line with studies in the breast (Maurer et al., 2009), lung (Shen etal., 2008), and colon cancer (Mampaey et al., 2015), wherein the 16p13gain was linked to poor prognosis. The minimal region of 16p13.3 gain(described in Choucair et al. 2012) spans 19 genes. PDPK1, encodingPDK1, is a likely driver of the gain but other genes involved in othertypes of cancer reside at this locus as well (Choucair et al., 2012). Ofthese, RAB26 is a Ras oncogene family member found to be upregulated innon-small cell lung carcinoma (Valk et al., 2010) and uveal melanoma(Marshall et al., 2007). Similarly, CCNF (G2-mitosis-specific cyclin-F)was reported to be overexpressed in breast and esophageal cancer,respectively (Tamoto et al., 2004; Williams et al., 2008). ABCA3, aknown drug efflux pump belonging to the p-gp family, is overexpressed inacute myeloid leukemia (AML) and different cancer cell lines. Notably,ABCA3 overexpression conferred drug resistance in these AML cases(Chapuy et al., 2008; Steinbach et al., 2006; Yasui et al., 2004). Therelevance of these genes in prostate cancer remains to be investigatedthrough functional studies.

One application for the 16p13.3 gain is a FISH biomarker to identifypatients requiring adjuvant or neoadjuvant therapies and to improvepretreatment prognostication given that accurate GS can be challengingto obtain on biopsies. Studies on needle biopsy cohort are desirable forits implementation in the presurgical setting. The spatial resolutionafforded by a histology-based assay such as FISH would facilitatesensitive assessment of individual cancer foci (Bishop et al., 2010;Gozzetti et al., 2000) in a context of tumor heterogeneity and bypassthe need for nucleic acid extraction from bulk tumor tissue required byseveral recently developed commercial assays based on gene expressionsignatures, such as Prolaris and Decipher (Cuzick et al., 2012; Irshadet al., 2013; Klein et al, 2014; Klein et al., 2016).

Taken together, the results support a role for 16p13.3 gain in prostatecancer progression and as a relevant prognostic biomarker. Incorporating16p13.3 gain status with routinely used clinicopathologic variablesallows for improvements to stratifying patients into differentprognostic groups.

Example IV—Materials and Methods for Pten Fish Analysis

Study population and tissue microarray. This study was done incompliance with the REMARK guidelines (McShane et al., 2005) andapproved by the Research Ethics Board of the McGill University HealthCentre (BDM-10-115) with the written informed consent of theparticipants. A set of 332 de-identified formalin-fixedparaffin-embedded (FFPE) radical prostatectomy specimens collectedbetween 1993 and 2008 at the McGill University Health Centre wererepresented on a tissue microarray by duplicate 1 mm cores extractedfrom the dominant tumor nodule. Dominant nodule was defined as generallythe largest nodule. In cases in which a smaller nodule was considered tobe prognostically more significant (higher grade or stage), this smallernodule was considered to be dominant.

TABLE 6 Clinicopathologic features of radical prostatectomy casesrepresented on the tissue microarray. Clinicopathologic variablesCategory n (%) Total number of cases n 332 Age (years) Median  61Min-max 43-73 Preoperative serum n^(a) 327 prostate-specific antigenMean (±SD) 8.66 (±8.27) (ng/ml) PSA ≤ 10 253 (77%) PSA > 10 74 (23%)Gleason grade groups at Group 1 (3 + 3) 70 (21%) surgery Group 2 (3 + 4)153 (46%) Group 3 (4 + 3) 78 (24%) Group 4 (8) 11 (3%) Group 5 (≥9) 20(6%) Pathological stage pT2 219 (66%) (T-stage) pT3a 91 (27%) pT3b 22(7%) Surgical margin status Positive 97 (29%) Follow-up (months) n^(a)297 Median (min-max) 116 (1-253) Biochemical recurrence n^(a) 297Positive 81 (27%) Distant matastases Positive 16/321^(a) (5%) ^(a)Valuesnot available for all the 332 cases (n noted for each variable)

The clinical information was retrieved from the medical charts and thepathological correlates were obtained after re-review of all the radicalprostatectomy cases by a single dedicated genitourinary pathologist. Thefinal Gleason grade was assigned according to the latest InternationalSociety of Urological Pathology/World Health Organizationrecommendations (Humphrey et al., 2016). The clinicopathologiccharacteristics of 303 of the 332 cases (see Example I) and those of theentire expanded cohort are summarized in Table 6. The mean preoperativeserum prostate-specific antigen level was 8.66 (±8.27) and thedistribution of Gleason grade group 1 (Gleason score 6), 2 (Gleasonscore 3+4), 3 (Gleason score 4+3), 4 (Gleason score 8), and 5 (Gleasonscore 9) was 21%, 46%, 24%, 3%, and 6%, respectively. Sixty-six percentof patients were at stage pT2 while 34% belonged to stage pT3. Patientsreceiving neoadjuvant hormone therapy (n=6) and cases with missing serumprostate-specific antigen data post-radical prostatectomy (n=15) werenot included in the biochemical recurrence analyses. Surgical failurecases (n=14), for which the serum prostate-specific antigen did not fallto undetectable levels post-radical prostatectomy, were also excludedfrom the biochemical recurrence analyses. No patient had receivedadjuvant radiation therapy after surgery. The primary endpoint of thestudy was biochemical recurrence and was defined by a serumprostate-specific antigen elevation of >0.2 ng/ml following radicalprostatectomy (27%). The recurrence-free interval was defined as thetime between the surgery date and the date of the firstprostate-specific antigen increase above 0.2 ng/ml. Patients withoutbiochemical recurrence event were censored at the last follow-up datewith prostate-specific antigen measurement. The median follow-up for thecohort was 116 months (1-253 months, min-max). Metastasis status wasevaluated and confirmed by imaging in patients with clinical symptoms(n=16). The metastasis-free interval was defined as the period betweenthe surgery date and the date of first metastasis detection and patientswithout signs/symptoms related to metastasis were censored at the lastfollow-up/prostate-specific antigen date. The CAPRA-S(Cancer of theProstate Risk Assessment Post-Surgical) score was calculated from thestatus of six clinicopathologic variables [preoperativeprostate-specific antigen, Gleason score, surgical margins,extracapsular extension, seminal vesicle invasion, lymph node invasion],and each patient was assigned to one of the three risk groups: low(0-2), intermediate (3-5), and high (≥6) according to Cooperberg et al.(2011). Of note, patients who did not undergo a lymph node dissectionwere considered to have negative lymph node for CAPRA-S scorecalculation as previously described (Punnen et al., 2014). Thechromosome 16p13.3 gain data of Examples I to III was used for thecombinatorial approach.

Fluorescence in situ hybridization (FISH). The BAC clone CTD-2557P6(BACPAC Resources Center, Oakland, Calif.) mapping to the PTEN gene onthe chromosome 10q23.3 region and commercially available CEP10 SpectrumGreen probe (CEP 10, Abbott Molecular, Abbott Park, Ill.), which spansthe 10p11.1-q11.1 centromeric region were used to perform dual-colorFISH on the 5 μm tissue microarray sections as described previously(Choucair, Ejdelman, et al., 2012). The CTD-2557P6 DNA was labeled withthe Spectrum Orange-dUTP (Enzo Life Science, Farmingdale, N.Y.) usingthe Nick Translation Reagent Kit (Abbott Molecular) as per the kitmanual.

FISH data analysis. To evaluate the PTEN copy number status, fluorescentsignals were counted in 100 non-overlapping interphase nuclei for eachcase (as identified on corresponding H&E) counterstained with ProLong®Gold antifade reagent with DAPI (Life Technology, CA), to delineatenuclei. The PTEN deletion was defined as ≥15% of tumor nuclei containingone or no PTEN locus signal and by the presence of two CEP10 signals aspreviously reported (Choucair, Ejdelman, et al., 2012). A tumor wasconsidered homozygous-deleted if ≥15% of tumor nuclei had no PTEN locussignals and two CEP10 signals. Images were acquired with an OlympusIX-81 inverted microscope at x96 magnification, using Image-Pro Plus 7.0software (Media Cybernetics, Rockville, Md.).

Statistical analysis. The association between copy number alterationsand the clinicopathologic indicators were assessed by Fisher's exacttest for categorical variables and unpaired t-test for continuousvariables. Kaplan-Meier curves were generated for biochemicalrecurrence-free and metastasis-free survival analysis. The log-rank testwas used to evaluate the significance of differences between thestratified survival functions. Cox regression analyses were used toevaluate univariate hazard ratios and multivariate Cox proportionalhazards regression analysis was performed to identify independentpredictors of biochemical recurrence. The C-index was calculated asdescribed by Harrell et al. (1996). Analyses were performed using SPSS,WinStet, and R (Version 3.3.2).

Example V—Results of Pten Fish Analysis

Association of PTEN deletion status with adverse clinical outcomepost-radical prostatectomy. The 10q23.3 (PTEN) deletion status wasassessed using dual-color FISH on 332 radical prostatectomy specimensrepresented on a tissue microarray. The clinicopathologic features ofthese patients are summarized in Table 6. The PTEN deletion status couldbe successfully assessed in 287 tumors arrayed out, of which 97 (34%)harbored a PTEN genomic deletion. The PTEN deletion status wasconsistent across duplicate TMA cores evaluated. Of the cases with PTENdeletion, 80 (28%) were hemizygous deleted while 17 (6%) harbored ahomozygous PTEN deletion (FIG. 6A to 6D). Of note, 15 out the 17 casesthat was identified as homozygous deleted for PTEN also harbored asignificant number of nuclei 5%) showing a hemizygous deletion withinthe tissue microarray core. Preliminary analysis indicated that thesecases with homozygous deletion were not different than cases harboringonly a hemizygous deletion in term of their association with adversepathology and poor outcome (not shown). Therefore hemizygous andhomozygous PTEN deletion cases were considered together as a singlegroup for the analyses presented in this report. As shown in Table 7,the PTEN deletion status was significantly associated with high Gleasongrade group (P=0.0001) and advanced pT-stage (P=0.001).

TABLE 7 Association of PTEN deletion status with clinicopathologicfeatures of aggressive prostate cancer. Total Clinicopathologic casesPTEN status variables n (%) No deletion Deletion P-value PTEN status 287190 (66%) 97 (34%) Gleason grade groups 287 0.0001 Group 1 (GS 3 + 3) 6152 (85%) 9 (15%) Group 2 (GS 3 + 4) 132 90 (68%) 42 (32%) Group 3 (GS4 + 3) 67 37 (55%) 30 (45%) Group 4 and 5 27 11 (41%) 16 (59%) (GS ≥ 8)Pathological T-stage 287 0.001 pT2 189 138 (73%) 51 (27%) pT3a 77 43(56%) 34 (44%) pT3b 21 9 (43%) 12 (57%) Preoperative prostate- 282 7.95(±7.60) 9.03 (±7.24) 0.25^(a) specific antigen (mean/±standarddeviation) Surgical margin status 287 0.30 Negative 200 136 (68%) 64(32%) Positive 87 54 (62%) 33 (38%) P-value calculated by Fisher exacttest Number of cases that could be assessed (n) noted for each variable^(a)Unpaired t-test

The prognostic significance of PTEN genomic status was first evaluatedusing biochemical recurrence as a surrogate primary endpointpost-radical prostatectomy. PTEN FISH status and completeprostate-specific antigen follow-up data were available for 256 radicalprostatectomy cases, out of which 69 (27%) experienced biochemicalrecurrence. The PTEN genomic deletion status emerged as a significantpredictor of early biochemical recurrence following radicalprostatectomy (log-rank P<0.0001; FIG. 7A) independent of the standardclinicopathologic prognostic indicators like Gleason grade group,pT-stage, preoperative prostate-specific antigen level, and surgicalmargin status in a multivariate Cox analysis (hazard ratio: 3.00, 95%confidence interval: 1.81-4.99; P<0.0001; Table 8, (A)).

Clinical significance of PTEN genomic deletion in low-intermediate riskprostate cancer patients. The ability of the PTEN deletion status wasassessed to predict biochemical recurrence (BCR) risk in a clinicallyrelevant subset of patients belonging to grade groups 1 and 2 (3+4)according to the latest International Society of UrologicalPathology/World Health Organization Gleason grading recommendations(Humphrey et al., 2016). The PTEN deletion status was significantlyassociated with biochemical recurrence in patients of grade group 1-2(3+4, log-rank, P<0.0001, FIG. 7B) including those that were also ofstage pT2 and with preoperative prostate-specific antigen ≤10 (log-rank,P=0.002, FIG. 7C). Furthermore, the PTEN deletion was significantlylinked to biochemical recurrence in a subgroup of grade group 2 (3+4,log-rank, P<0.0001, FIG. 7D) even with favorable stage pT2 andprostate-specific antigen ≤10 (log-rank, P=0.007, FIG. 7E). There was aninsufficient number of biochemical recurrence events (n=1) in gradegroup 1 (Gleason score 6) to allow subgroup analysis and the PTENdeletion status did not further stratify grade group 3-5 4+3, notshown). It was then assessed if the PTEN genomic deletion status couldfurther stratify the risk groups defined by the clinically validatedclinicopathologic CAPRA-S score to predict biochemical recurrencepostradical prostatectomy (Cooperberg et al., 2011). The multivariateanalysis showed that the PTEN deletion was a significant predictor ofbiochemical recurrence along CAPRA-S score risk groups (hazard ratio:2.84, 95% confidence interval: 1.75-4.63; P<0.0001, Table 8, (B)). PTENdeletion identified a subset of patients with a greater risk ofbiochemical recurrence among those of low and intermediate CAPRA-S scorerisk groups (FIGS. 7F and 7G), log-rank, P=0.0001 and P=0.0002,respectively). The association of PTEN deletion with bone or soft tissuemetastases (an important adverse secondary endpoint) was furtherevaluated. PTEN deletion status was indeed significantly associated withan increased risk of distant metastasis (log-rank P=0.001, FIG. 7H),further supporting its potential clinical utility as a marker ofprostate cancer progression.

TABLE 8 Univariate and multivariate Cox proportional hazard analysispredicting biochemical recurrence for PTEN deletion status adjusted for(A) standard clinicopathologic parameters and (B) CAPRA-S score.Univariate analysis Multivariate analysis Hazard ratio (95% Hazard ratio(95% Variables confidence interval) P-value confidence interval) P-value(A) Standard clinicopathologic parameters PTEN status (deleted vs. 3.47(2.14-5.63) <0.0001 3.00 (1.81-4.99) <0.0001 non-deleted) Preoperativeprostate-specific 1.06 (1.04-1.09) <0.0001 1.05 (1.01-1.08) 0.005antigen^(a) Gleason grade Group 3-5 (≥4 + 3) vs. 4.75 (2.91-7.78)<0.0001 2.60 (1.44-4.67) 0.001 group 1-2 (≤3 + 4) pT-stage (T3 vs. T2)3.32 (2.05-5.38) <0.0001 1.53 (0.87-2.67) 0.137 Surgical margin(positive vs. 2.30 (1.43-3.72) 0.001 1.88 (1.12-3.13) 0.016 negative)Age at surgery^(a) 1.02 (0.98-1.07) 0.256 0.98 (0.94-1.03) 0.494 (B)CAPRA-S score PTEN status (deleted vs. 3.47 (2.14-5.63) <0.0001 2.84(1.75-4.63) <0.0001 non-deleted) CAPRA-S risk Low (0-2) reference —<0.0001 — <0.0001 Intermediate (3-5) 3.40 (1.81-6.36) <0.0001 3.00(1.60-5.64) 0.001 High (≥6) 10.65 (5.42-20.91) <0.0001 8.95 (4.53-17.67)<0.0001 ^(a)Analyzed as a continuous variable; P-value: Wald test

Example VI—16P13.3 Gain and Pten Deletion Combination as PrognosticBiomarkers

Improved biochemical recurrence risk stratification upon combining16p13.3 gain with PTEN deletion. As discussed above, 16p13.3 genomicgain status is associated with aggressive clinicopathologic features ofprostate cancer (Choucair et al., 2012), as well as with poor clinicaloutcome. A set of 251 cases for which both 16p13.3 gain and PTENdeletion data were available was used for the combinatorial PTEN-16p13.3co-alteration analyses. It was first tested whether PTEN deletion statuscould further stratify patients without 16p13.3 gain. As shown in FIG.8A, cases with PTEN deletion have an increased risk of biochemicalrecurrence among this subgroup (log-rank, P<0.0001). Interestingly,amongst patients with no PTEN deletion, the 16p13.3 gain furtheridentified a subset of patients at high risk of recurrence (FIG. 8B,log-rank, P=0.001). Cases were then grouped based on their PTEN-16p13.3co-alteration status-(0) no PTEN deletion and no 16p13.3 gain, (1) PTENdeletion or 16p13.3 gain, and (2) PTEN deletion and 16p13.3 gain.Kaplan-Meier analysis demonstrated that the PTEN-16p13.3 co-alterationstatus further segregated prostate cancer cases in three distinctprognostic subgroups (logrank P<0.0001, FIG. 8C) stratified by thenumber of positive markers: the favorable prognostic group with noalterations in PTEN and 16p13.3, the intermediate prognostic group withone alteration in either PTEN or 16p13.3, and the worst prognostic groupwith two alterations (PTEN and 16p13.3). Moreover, in the multivariateCox analysis adjusted for standard prognostic indicators, thePTEN-16p13.3 co-alteration status remained significant and conferred thehighest risk of biochemical recurrence (hazard ratio: 4.18, 95%confidence interval: 1.82-9.59; P=0.001; Table 9, (A)). Similarly, aPTEN deletion along a 16p13.3 gain increased the risk of recurrencesignificantly even after adjusting for the CAPRA-S score risk groups(hazard ratio: 4.70, 95% confidence interval: 2.12-10.42, P<0.0001,Table 9, (B)). To estimate the potential prognostic benefit of assessingboth genomic alterations, the C-index was calculated for each of PTENdeletion and 16p13.3 gain alone and in combination using biochemicalrecurrence as an endpoint in Cox model. The C-index was higher by usingboth alterations than 16p13.3 gain or PTEN deletion alone (0.69 vs. 0.62and 0.63, respectively) and each of these alterations improved theC-index of the CAPRA-S score reaching a maximum when both were included(0.78, FIG. 8D).

TABLE 9 Univariate and multivariate Cox proportional hazard analysispredicting biochemical recurrence for the PTEN-16p13 co-alterationstatus adjusted for (A) standard clinicopathologic parameters and (B)CAPRA-S score. Unicariate analysis Univariate analysis Hazard ratio (95%Hazard ratio (95% Variables confidence interval) P-value confidenceinterval) P-value (A) Standard clinicopathologic parameters PTEN-16p13co-alteration status No PTEN and No 16p13 — <0.0001 — 0.03 (reference)PTEN or 16p13 3.93 (1.88-8.25) <0.0001 2.90 (1.35-6.22) 0.06 PTEN and16p13 6.88 (3.14-15.08) <0.0001 4.18 (1.82-9.59) 0.001 Preoperativeprostate- 1.08 (1.05-1.10) <0.0001 1.05 (1.02-1.08) 0.002 specificantigen^(a) Gleason grade Group 3-5 (≥4 + 3) vs. 4.70 (2.79-7.90)<0.0001 2.16 (1.14-4.12) 0.019 group 1-2 (≤3 + 4) pT-stage (T3 vs. T2)3.29 (1.98-5.48) <0.0001 1.46 (0.80-2.67) 0.217 Surgical margin(positive vs 2.25 (1.37-3.72) 0.002 1.50 (0.87-2.57) 0.143 negative) Ageat surgery^(a) 1.03 (0.98-1.07) 0.263 0.98 (0.96-1.05) 0.924 (B) CAPRA-Sscore PTEN-16p13 co-alteration status No PTEN and no 16p13 — <0.0001 —0.001 (reference) PTEN or 16p13 3.93 (1.88-8.25) <0.0001 3.55(1.69-7.46) 0.001 PTEN and 16p13 6.88 (3.14-15.08) <0.0001 4.70(2.12-10.42) <0.0001 CAPRA-S risk Low (0-2) reference — <0.0001 —<0.0001 Intermediate (3-5) 3.21 (1.66-6.19) 0.001 2.70 (1.40-5.23) 0.003High (≥6) 9.25 (4.58-18.69) <0.0001 7.34 (3.57-15.07) <0.0001^(a)Analyzed as a continuous variable; P-value: Wald test

In this example, the association of PTEN deletion with poor outcome inprostate cancer was confirmed and its potential at further stratifyinglow-intermediate risk patients treated by radical prostatectomy wasdemonstrated. In addition, it was shown that its prognostic value can beimproved by considering the gain of 16p13.3. PTEN deletion was detectedby FISH in 34% of the 287 radical prostatectomy specimens examined, afrequency falling within the range of 17-42% reported by otherpreviously published studies using FISH and including over hundredsamples (Krohn et al., 2012; Qu et al., 2016; Troyer et al., 2015;Yoshimoto et al., 2007; Bismar et al., 2011; Han et al., 2009). Themajority of deletions observed were hemizygous (28% vs. 6% ofhomozygous) in agreement with most of the previous publications onradical prostatectomy cases. In contrast, Krohn et al. (2012) reported12% of homozygous and 8% of hemizygous deletion in their cohort whileTroyer et al. (2015) observed 9% homozygous and 9% hemizygous deletionin their samples. The variation in frequency of PTEN deletion and inproportion of hemizygous vs. homozygous deletion observed among thestudies possibly reflects differences of cohort sizes andclinicopathologic features, but also likely differences in tissuepreparation and FISH scoring method. The presence of homozygous-deletedand hemizygous deleted nuclei in most of tumor classified as PTENhomozygous-deleted in this study likely reflect intratumoralheterogeneity and possibly disease progression.

Supporting a role for PTEN alteration in prostate cancer progression,this study showed that its deletion was significantly associated withthe aggressive clinicopathologic features of high Gleason grade groupand advanced surgical stage pT3, a finding consistent with previousreports of PTEN FISH on large radical prostatectomy sets (Krohn et al.,2012; Troyer et al., 2015). In agreement with prior report on a separatesample set (Choucair, Ejdelman, et al., 2012) as well as with previousstudies of other groups (Krohn et al., 2012; Qu et al., 2016; Troyer etal., 2015; Yoshimoto et al., 2007), it was also shown that PTEN deletionassessed by FISH was associated with biochemical recurrence afterradical prostatectomy. Moreover, the prognostic value of the deletionwas independent of standard clinicopathologic markers. In this study,homozygous deletion was not associated with a higher risk of biochemicalrecurrence than hemizygous deletion in agreement with the report ofKrohn et al. (2012), but in contrast to Yoshimoto et al. (2007) andTroyer et al. (2015). Present data indicate that the loss of one copywas sufficient to increase significantly the risk of biochemicalrecurrence, which is consistent with PTEN haploinsufficiencydemonstrated in prostate cancer animal models (Kwabi-Addo et al., 2001).It is also possible that the second allele has been inactivated byalternative mechanisms (Whang et al., 1998), which was not investigatedin this study. Interestingly, the PTEN deletion status could furtherstratify patients of low-intermediate risk grade group 1-2 (3+4), pT2,and prostate-specific antigen <10, a finding not reported in previousstudies. The revised Gleason scoring system applied to the presentcohort offers more refinement over previous iterations by splittingGleason score 7 into two groups of distinct prognoses: grade group 2(3+4) and grade group 3 (4+3). While the grade group 2 has the bestoutcome, it was further stratified by PTEN FISH. Since the percentage ofGleason pattern 4 was not recorded in this cohort, it is unclear if andhow it would correlate with the PTEN status. Similarly, PTEN FISH wasable to sub-classify cases belonging to low and intermediate CAPRA-Srisk groups, thus emphasizing the potential complementary role of thismarker to clinicopathologic assessment for outcome prediction.

PTEN deletions detected by FISH are known to be enriched in metastaticas compared to primary prostate tumors (Han et al., 2009; Qu et al.,2013). One PTEN FISH study reported metastasis outcome on radicalprostatectomy specimens, but did not find any significant associationwith PTEN deletion (Troyer et al., 2015). Here, it was shown thatpatients with a PTEN deletion in their radical prostatectomy sample wereat a higher risk of experiencing distant metastases. Present results arein agreement with Lotan et al. (2011) who used a selected high-riskradical prostatectomy cohort (all patients experienced a biochemicalrecurrence) to demonstrate that the loss of PTEN protein expression wasassociated with a shorter time to distant metastasis. While furthervalidation on different cohorts is needed, present findings highlightthe potential of PTEN deletion as a marker of disease progression toadvanced metastatic disease.

As discussed above in Examples II and III, 16p13.3 gain is associatedwith aggressive clinicopathologic features of prostate cancer as well asan increased risk of biochemical recurrence and distant metastases inthe same radical prostatectomy specimens surveyed here for PTENdeletion. Moreover, the 16p13.3 gain status improved the stratificationof patients with intermediate and high risk of disease progression basedon their CAPRA-S score. The analysis of the combined data presented hereexemplified the advantages of considering both PTEN and 16p13.3 CNAs forbiochemical recurrence risk stratification. Cases that were negative forPTEN deletion were further stratified by the 16p13.3 gain status andvice versa, thus allowing the identification of patients that have areduced risk of biochemical recurrence. A maximum risk of biochemicalrecurrence was reached for patients whose tumors harbored both PTENdeletion and 16p13.3 gain. The advantage of this combinatorial approachwas further evidenced by an increase of the C-index, which reached itsmaximum when coupled with the CAPRA-S score risk groups. Interestingly,the PTEN deletion status identified patients of CAPRA-S low-risk groupwho have an increased risk of biochemical recurrence, while the 16p13.3gain status alone did not further stratify the low-risk group. Theseresults are in agreement with previous studies showing that combinationsof genomic features such as gene expression changes and copy numberalterations, including PTEN deletion status, can add prognosticinformation to the CAPRA-S score (Cooperberg et al., 2015; Lennartz etal., 2016). Owing to the relatively small number of secondary adverseevents like metastases and prostate cancer-specific deaths in thiscohort, additional future studies can be performed focusing on assessingthe clinical significance of this PTEN-16p13.3 co-alteration status withrespect to these adverse clinical end-points in large independentcohorts with long clinical follow-up.

Given the enhanced risk of biochemical recurrence associated withco-alteration of PTEN and 16p13.3, patients with such tumors may benefitfrom adjuvant treatments. In prostate cancer, the usefulness of adjuvantsystemic therapy such as chemotherapy postsurgery remains to beestablished (Pignot et al., 2018). The lack of demonstratedeffectiveness may be explained in part by differences of tumor biologyamong patients. Molecular biomarkers such as PTEN/16p13.3 FISH assistsin patient selection and thus improve the success of future clinicaltrials. The assessment of these markers retrospectively in samples ofpatients who have received adjuvant therapy may provide additional datato support this hypothesis.

PTEN inactivation occurs predominantly via genomic deletion at 10q23(Cancer Genome Atlas Research Network, 2015; Cairns et al, 1997), whichresults in increased levels of phosphatidylinositol [3-5]-trisphosphate(PIP3). PIP3 triggers the phosphorylation and activation of AKT by PDK1leading to the stimulation of pathways related to cell growth andsurvival (Toren et al., 2014). PDPK1 encoding PDK1 at 16p13.3 wasidentified as a potential driver of the gain and found that PDK1stimulates prostate cancer cell migration in vitro (Choucair et al.,2012). Increasing data from the literature indicate that PDK1 not onlyphosphorylates AKT, but is also directly involved in cell invasion andmigration (Gagliardi et al., 2015). It is possible that anoverexpression of PDK1 resulting from 16p13.3 gain would potentializethe PIK3/AKT pathway activation and/or provide additional advantagesrelevant to tumor progression such as an increased cell motility leadingto tumor cell spreading beyond the prostate, which may explain theaugmented risk of tumor recurrence associated with both PTEN and 16p13.3copy number alterations. Assessment of the in situ expression andactivation status of the proteins involved in the PIK3/AKT pathway aswell as further work on animal models are warranted to elucidate therole of PDK1 in PCa and validate this hypothesis.

Genomic instability, as reflected by the percentage of tumor genomeharboring copy number alterations, has been shown to be associated withadverse outcome (Hieronymus et al., 2014; Lalonde et al., 2014). It isthus possible that the higher risk of biochemical recurrence associatedwith PTEN-16p13.3 co-alteration reflects an increased genomicinstability. PTEN has been shown to contribute to genomic instabilityleading to aggressive prostate cancer in animal models (Hubbard et al.,2016). In previous CAN analysis, 8q24 gain (MYC) and 16q23 deletionalong PTEN deletion and 16p13 gain was identified as the four mostcommon alterations enriched in lymph node metastases (Lapointe et al.,2007). It has been shown that the co-deletion of PTEN and 16q23 wasassociated with poor outcome after radical prostatectomy (Kluth et al.,2015). These other key copy number alterations may provide additionalprognostic value, in particular MYC that synergizes with PTEN for tumorinitiation and progression in animal models (Hubbard et al., 2016).

One use for such a panel of copy number alterations would be as FISHbiomarkers on diagnostic biopsies to identify more effectively patientssuitable for active surveillance, ultimately improving the pretreatmentprognostication given that accurate Gleason grade on biopsies can bechallenging to obtain. PTEN deletion detected in prostate needlebiopsies of Gleason score 6 (grade group 1) has been shown to beassociated with upgrading to Gleason score 7+(grade group 2 and up) atradical prostatectomy (Picanco-Albuquerque et al., 2016), which suggeststhat molecular biomarkers may overcome some of the limitationsassociated with the standard histopathologic evaluation of biopsyspecimens. The improved patient risk stratification afforded bycombining PTEN/16p13.3 may also be applicable in biopsy specimens froman active surveillance cohort.

FISH is regarded as the gold standard for the assessment of copy numberalterations in tissue specimens owing to its ability to delineatespecific genomic events with a spatial resolution facilitating asensitive evaluation of individual cancer foci at a single-cell level(Bishop et al., 2010; Gozzetti et al., 2000). Assessing the expressionof encoded proteins by immunohistochemistry is considered as analternative approach to capture the prognostic value associated withcopy number alterations. Previous PTEN immunohistochemistry assaysapplied to large cohorts have yielded variable results in terms ofoutcome prediction and correlation with FISH (Krohn et al., 2012; Cuzicket al., 2013). An improved PTEN immunohistochemistry assay was appliedrecently to one of the cohorts previously analyzed byimmunohistochemistry and FISH mentioned above (Krohn et al., 2012) andthe authors reported a sensitivity of 83% and 67% to respectively detecthomozygous and hemizygous deletion (Lotan et al., 2017). While PTENprotein loss assessed by immunohistochemistry was associated withincreased risk of biochemical recurrence, a further risk stratificationwas achieved by combining FISH with immunohistochemistry results,substantiating the enhanced value of PTEN FISH in outcome prediction.Future studies comparing both FISH and immunohistochemistry onadditional cohorts, including present cohort, would be important toconfirm these observations.

In summary, the results of this study support the prognostic value ofPTEN deletion in prostate cancer, which can be further improved incombination with 16p13.3 gain status, suggesting that these genomicalterations may cooperatively contribute to prostate cancer progression.DNA copy number analysis of PTEN and 16p13.3 could be of importantclinical value particularly for preoperative risk assessment of theclinically most challenging group of low-grade and intermediate-gradeprostate cancer.

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What is claimed is:
 1. A method of prognosing a subject with prostatecancer, the method comprising: (a) providing a first sample from theprostate from the subject suspected of comprising cancer cells; (b)contacting the first sample with a first probe capable of specificallyrecognizing a 16p13.3 chromosome region, wherein the first probe isassociated with a first label; (c) contacting the first sample with afirst reference probe capable of specifically recognizing a referenceregion of chromosome 16, wherein the first reference probe is associatedwith a first reference label; (d) detecting the signal from the firstlabel and from the first reference label; and (e) classifying the cancerrisk of the subject based on the presence or absence of a 16p13.3 gain,wherein the 16p13.3 gain is detected if the signal from the first labelof the first probe is greater than the signal from the first referencelabel of the first reference probe.
 2. The method of claim 1, furthercomprising: (f) providing a second sample from the prostate from thesubject suspected of comprising cancer cells; (g) contacting the secondsample with a second probe capable of specifically recognizing a 10q23.3(PTEN) chromosome region, wherein the second probe is associated with asecond label; (h) contacting the second sample with a second referenceprobe capable of specifically recognizing a reference region ofchromosome 10, wherein the second reference probe is associated with asecond reference label; (i) detecting the signal from the second labeland from the second reference label; and (j) classifying the cancer riskof the subject based on the presence or absence of a PTEN deletion,wherein the PTEN deletion is detected if the signal from the secondlabel is less than the signal from the second reference label.
 3. Themethod of claim 1, wherein the sample is a tumor sample.
 4. The methodof claim 2, wherein the first sample is the second sample.
 5. The methodof claim 1, wherein the subject presents low to intermediate riskclinical features.
 6. The method of claim 1, wherein the first probe isderived from a RP11-20123 DNA clone, variants thereof, fragmentsthereof, or complements thereof.
 7. The method of claim 1, wherein thefirst probe is capable of hybridizing to one or more genes of the16p13.3 chromosome region comprising PKD1, RAB26, TRAF7, CASKIN1, MLST8,PGP, E4F1, DNASE1L2, ECI1, RNPS1, ABCA3, ABCA17A, CCNF, NTN3, TBC1D24,ATP6VOC, AMDHD2, CEMP1, PDPK1, variants thereof, fragments thereof, orcomplements thereof.
 8. The method of claim 1, wherein the firstreference probe is capable of specifically recognizing a 16qhcentromeric region.
 9. The method of claim 1, wherein the firstreference probe is a derived from a pHuR-195 DNA clone, variantsthereof, fragments thereof, or complements thereof.
 10. The method ofclaim 2, wherein the second probe is derived from a CTD-2557P6 DNAclone, variants thereof, fragments thereof, or complements thereof. 11.The method of claim 2, wherein the second reference label is associatedwith a second reference probe capable of specifically recognizing a10p11.1-q11.1 centromeric region.
 12. The method of claim 2, wherein thesecond reference probe is a CEP10 probe, variants thereof, fragmentsthereof, or complements thereof.
 13. The method of claim 1, wherein thelabel is a fluorescent label.
 14. The method of claim 13, comprisingdetecting the fluorescent labels with fluorescent in situ hybridization(FISH) method.
 15. The method of claim 1, further comprising treatingthe subject with an adjuvant or a neoadjuvant therapy if the subject ischaracterized as being associated with poor prognosis.
 16. The method ofclaim 15, wherein the adjuvant or neoadjuvant therapy comprises surgery,radiation therapy, hormonotherapy and/or chemotherapy.
 17. The method ofclaim 1, wherein the subject is a human.
 18. The method of claim 1,wherein the subject has previously had a radical prostatectomy.
 19. Themethod of claim 1, wherein the sample is from a resected tissue or froma biopsy.